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QP44.P831903      Experiments  for  stud    | 


EXPERIMENTS 


FOR    STl'DEKTS    IN    THE 

HARVARD   MEDICAL   SCHOOL 


STfu'tti  Etrition 


By  W.   T.   PORTER 


:ap 


COLUMBIA  UNivPRPtTY 

DEPARTMENT  OF  PHrSlfHOHY 

College  of  Physicians  and  Surgeons 
437  west  fifty  ninth  stkeet 

NEW  YORK 


THE    UNIVERSITY    PRESS 

Camijrtfigr,  fftass. 
1903 


Columbia  <18ntoer*ftp 

College  of  $'op£trians  anb  iburgeons 
Hibrarp 


Digitized  by  the  Internet  Archive 

in  2010  with  funding  from 
Columbia  University  Libraries 


http://www.archive.org/details/experimentsforstOOport 


EXPERIMENTS 


FOR    STCDEXTS    IX    THE 


HARVARD    MEDICAL   SCHOOL 


NOTICE 

The  experiments  performed  by  Harvard  med- 
ical students  are  contained  in  the  following 
publications : 

1.  An  Introduction  to  Physiology,  Parts  I  and 
II,  containing  experiments  in  general  physiology,  in- 
cluding muscle  and  nerve,  and  in  the  circulation  of 
the  blood. 

2.  An  Introduction  to  Physiology,  Part  IV, 
Physiological  Optics. 

3.  Experiments  for  Students  in  the  Harvard 
Medical  School,  Third  Edition,  containing  experi- 
ments upon  the  central  nervous  system,  skin,  general 
sensations,  taste,  vision,  fermentation,  digestion,  blood, 
respiration,  and  metabolism. 

Additional  experiments  will  be  added  as  rapidly  as 
possible. 


EXPERIMENTS 


FOR    STUDENTS    IN    THE 


HARVARD  MEDICAL  SCHOOL 


QftixXi  lErjittcm 


By  W.   T.   PORTER 


THE    UNIVERSITY    PRESS 

Cambridge,  fflass. 

1903 


4\ 

no3 


Copyright,  1903 
By   W.  T.  Porter 


cry 


CONTEXTS 


The  Central  Nervous  System 

Simple  Reflex  Actions 1 

The  spinal  cord  a  seat  of  simple  reflexes  —  Influence  of 
afferent  impulses  on  reflex  action  —  Threshold  \alue 
lower  in  end  organ  than  in  nerve-trunk  —  Summation 
of  afferent  impulses — SegnYental  arrangement  of  rellex 
apparatus  —  Reflexes  in  man. 

Tendon  Reflexes       6 

Knee  jerk  —  Ankle  jerk  —  Gower's  experiment. 
Effect  of  Strychnine  on  Reflex  Action        ...        8 

Complex  Co-ordinated  Reflexes 8 

Removal  of  cerebral  hemispheres  —  Posture,  etc.  —  Bal- 
ancing experiment  —  Retinal  retire  —  Croak  reflex. 

Apparent  Purpose;  in  Reflex  Action 12 

Reflex  and  Reaction  Time 13 

Reflex  time  —  Reaction  time  —  Reaction  time  with  choice. 

Inhibition  of  Reflexes         15 

Through  peripheral  afferent  nerves  —  Through  central 
afferent  paths  ;  the  optic  lobes. 

The  Roots  of  Spinal  Nerves 17 

Lttdwig's  demonstration  —  Localization  of  movements  at 
different  levels  of  the  spinal  cord. 


VI  CONTENTS 

Distribution  of  Sensory  Spinal  Nerves     ....      19 

Muscular  Tonus 20 

Brondgeest's  experiment. 


II 

The  Skin 

Sensations  of  Temperature .      21 

Hot  and  cold  spots  —  Outline  —  Mechanical  stimulation 
—  Chemical  stimulation  —  Electrical  stimulation  — Tern 
perature  after-sensation  —  Balance  between  loss  and  gaiii 
of  heat  —  Fatigue  —  Relation  of  stimulated  area  to  sen- 
sation —  Perception  of  difference  —  Relatively  insensitive 
regions. 

Sensations  of  Pressure 24 

Pressure  spots  —  Threshold  value  —  Touch  discrimina- 
tion —  Weber's  law  —  After-sensation  of  pressure  — 
Temperature  and  pressure  —  Touch  illusion  ;  Aristotle's 
experiment. 


Ill 

General  Sensations 

Tickle 29 

Irradiation  —  After  image  —  Topography  —  Summation  — 
Fatigue. 

Pain 30 

Threshold  value — Latent  period — Summation  —  Topog- 
raphy —  Individual  variation  —Temperature  stimuli. 

Motor  Sensations 31 

Judgment  of  weight  —  Sensation  of  effort  —  Sensation  of 
motion. 


CONTEXTS  VU 


IV 


Taste 

Threshold  value  —  Topography  —  Relation  of  taste  to  area 

stimulated  ■ —  Electrical  stimulation 32 


Vision 

Mapping  the  blind  spot  —  Yellow  spot  —  Field  of  vision. 
Color  Blindness 35 

Method  of  examination  and  diagnosis. 

VI 

Fermentation 

Specific  Action 38 

Conversion  of  starch  to  sugar  by  germinating  barley  —  Con- 
version of  starch  to  sugar  by  salivary  diastase  (ptyalin)  — 
Extraction  of  diastase  from  germinating  barley  —  Specific 
action  of  ferments. 

Proteid  Digestion  by  Pepsin 41 

Gastric  digestion  of  cooked  beef  and  bread  —  Artificial 
gastric  juice  —  Digestion  with  artificial  gastric  juice  — 
Extraction  of  pepsin  —  Change  of  proteid  to  peptone  by 
pepsin. 

Splitting  of  Casein  by  Renntn 44 

Rennin  extract  —  Separation   of  rennin  —  Precipitation   of 

casein  —  Experiments  of  Arthus  and  Pages. 
Precipitation  of  Fibrin  by  Fibrin  Ferment  ...       48 
Buchanan's  experiment  —  Extraction  of  fibrin  ferment  — 

Extraction  of  fibrinogen  —  Precipitation  of  fibrinogen  by 

fibrin  ferment. 

Ammoniacal  Fermentation  of  Urea  by  Urease  .     .       50 
Extraction  of  urease. 


Vlll  CONTENTS 

Splitting  and  Synthesis  of  Fats 54 

Chemistry  of  fats  and  soaps  —  Splitting  of  fats  by  the  pan- 
creatic juice  —  Preparation  of  neutral  fat  —  The  emulsion 
test  for  fatty  acid  —  Extraction  of  lipase  —  Hydrolysis 
of  ethyl  butyrate  by  lipase  —  Syuthesis  of  neutral  fat 
by  lipase. 

Immunity 67 

Ehrlich's  ricin  experiments  —  Ricin  antitoxine  —  Theory 
of  immunity. 

HAEMO LYTIC   AND    BACTERIOLYTIC    FERMENTS    ....         76 
Bordet's  experiments. 

Oxidizing  Ferments 79 

Schonbein's  experiment  —  Further  oxidations  by  animal 
tissues  —  Oxidation  by  nucleo-proteid  —  Oxidation  about 
the  nucleus  —  Glycolysis  in  blood —Oxidation  not  de- 
pendent on  living  cells  of  blood  —  Relation  of  glycolysis 
to  the  pancreas  and  the  lymph— Glycolytic  ferment  of 
pancreas. 

Alcoholic  Fermentation 87 

The  yeast  plant  —  Chemical  relations  of  carbohydrates. 


VII 

Blood 

Specific  Gravity  . 94 

Drawing  the  blood  —  Determination  of  specific  gravity- 
Counting  the  red  corpuscles  —  Counting  the  white  cor- 
puscles. 

Estimation  of  Haemoglobin 100 

Hoppe-Seyler's  method  (modified). 

Haemorrhage  and  Regeneration  .......     101 

Alkalinity 102 

Zuntz-Loewy-Engel  method. 

Coagulation  Time 104 


CONTENTS  IX 

VIII 

Respiration 

Chemistry  of  Respiration 104 

Estimation  of  oxygen,  carbon  dioxide,  and  water. 

Mechanics  of  Respiration 106 

Artificial  scheme  —  Inspiration  —  Expiration  —  Normal 
respiration  —  Forced  respiration  —  Obstructed  air  pas- 
sages —  Asphyxia  —  Coughing :  sneezing  —  Hiccough  — 
Perforation  of  the  pleura. 


EXPERIMENTS 

FOR   STUDENTS    IX    THE 

HARVARD   MEDICAL   SCHOOL 

I 
THE   CENTRAL   NERVOUS    SYSTEM 

Simple  Reflex  Actions 

The  Spinal  Cord  a  Seat  of  Simple  Reflexes.  — 
1.  By  means  of  a  hook  or  thread  passed  through 
the  lower  jaw  suspend  vertically  a  frog  the  brain 
of  which  has  been  destroyed  with  the  seeker  ;  the 
legs  must  not  touch  the  table.  Pinch  a  toe  with 
the  forceps. 

The  leg  will  be  drawn  up. 

A  stimulus  to  the  skin  has  caused  the  con- 
traction of  muscles.  The  afferent  impulse  set 
going  by  the  sensory  stimulus  is  changed  into 
a  motor  efferent  impulse.  This  is  an  example  of 
reflex  action. 

2.  Destroy  the  spinal  cord  with  the  seeker. 
Stimulate  the  skin  of  the  right  leg  electrically 
and  mechanically. 


2        THE  CENTRAL  NERVOUS  SYSTEM 

In  no  case  will  the  sensory  stimulus  call  forth 
the  reflex  contraction  of  a  skeletal  muscle.  Yet 
the  nerves  coming  from  the  skin  and  going 
to  the  muscles  are  still  intact.  Only  the  spinal 
cord  has  been  destroyed. 

The  conversion  of  sensory  into  motor  impulses 
for  skeletal  muscles  is  a  function  of  the  central 
nervous  system. 

Influence  of  Afferent  Impulses  on  Reflex  Action. — 
Destroy  the  brain  of  a  strong  frog  with  the  seeker. 
Gently  pinch  a  toe  of  the  right  foot. 

Only  the  right  leg  will  be  drawn  up. 

Pinch  a  toe  of  the  left  foot. 

Only  the  left  foot  will  be  drawn  up. 

Pinch  a  finger. 

Only  the  corresponding  arm  will  move. 

Pinch  the  whole  foot  sharply. 

More  extended  movements  will  be  made. 

The  character  and  location  of  the  stimulus 
affect  the  resulting  contraction. 

Threshold  Value  Lower  in  End  Organ  than  in 
Nerve-Trunk. — 1.  Carefully  expose  the  sciatic 
nerve.  Determine  the  least  strength  of  tetanizing 
current  that  will  cause  a  crossed  reflex  when 
applied  to  the  skin  of  the  foot.  Now  apply  the 
same  stimulus  to  the  trunk  of  the  nerve. 

As  a  rule,  the  intensity  required  to  produce 
reflex  action  is  less  when  the  stimulus  is  applied 


SIMPLE    KEFLEX    ACTIONS  6 

to  the  peripheral  endings  of  the  sensory  nerves 
than  when  the  nerve-trunks  are  stimulated. 

2.  Divide  the  skin  over  the  back  in  the  median 
line.  Raise  the  skin  on  one  side  until  the  small 
nerves  which  pass  across  the  dorsal  lymph  sac  to 
innervate  the  skin  come  into  view.  Sever  from 
the  surrounding  skin  a  piece  about  one  centi- 
metre square  containing  the  endings  of  one  of 
the  nerves.  Let  the  isolated  piece  with  its  nerve 
endings  remain  connected  with  the  body  only  by 
the  trunk  of  the  nerve.  As  before,  determine 
the  least  strength  of  tetanizing  current  that  will 
cause  a  reflex  movement  when  applied  to  the 
nerve-endings  in  the  skin  and  to  the  nerve-trunk 
respectively.  * 

The  threshold  value  for  reflex  action  will  again 
be  found  lower  in  the  nerve-endings  than  in  the 
nerve-trunk. 

Summation  of  Afferent  Impulses. —  Pass  two 
fine  copper  wires  about  the  -frog's  foot  a  centi- 
metre apart  and  connect  them  with  the  secondary 
coil.  Connect  the  primary  coil  through  a  simple 
key  with  a  dry  cell.  Stimulate  with  regularly 
repeated  make  induction  currents  of  such  strength 
that  single  stimuli  cause  no  reflex  contraction. 

Summation  of  the  subminimal  stimuli  will 
finally  cause  reflex  contraction. 

Determine   that  the  number  of  stimuli  neces- 


4        THE  CENTRAL  NERVOUS  SYSTEM 

saryto  produce  a  reflex  becomes  smaller  when  (1) 
the  strength  of  the  induction  currents  is  increased, 
and  (2)  when  the  interval  between  the  stimuli  is 
lessened. 

Segmental  Arrangement  of  Reflex  Apparatus.  — 
1.  Gently  pass  the  seeker  over  the  abdominal 
walls  on  one  side. 

The  muscles  in  that  region  only  will  twitch. 

Eepeat  the  stimulus,  but  use  a  stronger 
pressure. 

The  area  contracting  will  increase  in  extent 
approximately  in  proportion  to  the  increase  in 
the  stimulus.  The  afferent  nerves  from  any  one 
region  are  more  closely  related  to  the  efferent 
nerves  of  that  same  region  than  to  those  of  other 
regions.  The  fact  that  both  afferent  and  efferent 
fibres  spring  from  the  cord  at  the  same  level 
suggests  that  their  nerve  C3lls  lie  also  at  approxi- 
mately the  same  level.  On  increasing  the  stim- 
ulus the  afferent  impulse  spreads  from  segment 
to  segment  of  the  cord.  Further  evidence  of  the 
segmental  arrangement  will  be  gained  by  the 
following  experiment. 

2.  With  a  clean,  sharp  knife  make  transverse 
sections  of  the  spinal  cord,  beginning  in  the  cer- 
vical region.  A  short  time  after  each  section 
test  the  reflexes  from  the  hind  limb  by  mechani- 
cal stimulation. 


SIMPLE    REFLEX    ACTIONS  5 

Note  the  level  below  which  no  section  can  be 
made  without  rendering  the  reflex  impossible. 
The  nerve  cells  concerned  in  this  reflex  lie  on 
the  caudal  side  of  this  line. 

Now  in  a  second  frog  make  transverse  sections, 
beginning  at  the  caudal  end  of  the  cord,  and  test 
the  reflexes  as  before,  until  the  level  is  reached 
beyond  which  a  section  will  destroy  the  reflex. 

Observe  that  the  portion  of  the  cord  comprised 
between  the  two  levels  determined  forms  a  seg- 
ment which  contains  the  central  apparatus  con- 
cerned in  the  reflex  studied. 

Reflexes  in  Man.  —  1.  From  the  Skin.  —  Eub 
the  plantar  surface  of  the  foot  gently  with  some 
hard  object. 

The  foot  will  be  retracted  reflexly. 

Similar  results  may  be  obtained  by  rubbing 
the  skin  of  the  inside  of  the  thigh,  which  will 
cause  contraction  of  the  cremaster  muscles ;  or  by 
rubbing  the  skin  of  the  abdomen,  which  will  be 
followed  by  contraction  of  the  abdominal  muscles. 

These  reflexes  are  of  importance  in  clinical 
diagnosis  because  by  means  of  them  the  seat  of  a 
diseased  area  in  the  central  nervous  system  may 
sometimes  be  defined,  since  the  reflex  depends  on 
the  integrity  of  the  corresponding  reflex  arc. 

2.  Cornea  Reflex.  —  Touch  the  cornea  gently 
with  a  thread. 


6        THE  CENTRAL  NERVOUS  SYSTEM 

The  eye  will  be  closed  involuntarily. 

3.  Throat  Reflex.  —  Touch  the  posterior  wall 
of  the  throat. 

The  movements  of  swallowing  will  usually 
follow. 

4.  Pupil  Reflexes ;  Light  Reflex.  —  Close  one 
eye  for  several  seconds,  then  open  it  quickly. 

Note  the  contraction  of  the  pupil. 

5.  Consensual  Reflex.  —  Close  one  eye  as  before, 
but  watch  the  pupil  of  the  other  eye  when  the 
first  is  opened  again. 

The  pupil  will  contract. 

6.  Accommodation  Reflexes. —  Look  alternately 
at  a  near  and  a  far  object.  The  pupil  will  con- 
tract when  the  eye  adjusts  itself  to  see  the  near 
object. 

Tendon  Eeflexes 

Knee  Jerk. — Sit  in  such  a  position  that  the 
knee  is  bent  at  a  right  angle,  and  the  foot  hangs 
free.  Let  an  assistant  strike  the  patellar  liga- 
ment with  the  side  of  the  hand. 

Note  the  sudden  contraction  of  the  extensors 
of  the  thigh,  the  so-called  knee  jerk. 

Flex  the  knee  at  different  angles  and  deter- 
mine in  which  position  the  resulting  contraction 
is  greatest. 

Knee  jerk  can  be  obtained  only  within  certain 
limits  of  extension. 


TENDON    REFLEXES  7 

Let  the  subject  immediately  before  the  stimu- 
lus is  applied  forcibly  contract  some  other  group 
of  muscles  ;  clench  the  hand,  for  example. 

The  knee  jerk  is  reinforced. 

Ankle  Jerk.  —  Bend  the  foot  at  ri<?ht  angles  to 
the  leg,  and  strike  the  tendo  Achillis.  The  ex- 
perimenter should  hold  the  end  of  the  foot  in  his 
left  hand.' 

Contraction  of  the  gastrocnemius  muscle  will 
be  observed. 

Gower's  Experiment.  —  Strike  the  side  of  the 
tendo  Achillis. 

A  contraction  will  result. 

Support  the  other  side  of  the  tendon  so  that 
the  gastrocnemius  muscle  will  not  be  stretched 
by  the  blow.     Eepeat  the  experiment. 

No  contraction  follows.  The  tendon  jerk  re- 
quires for  its  production  a  rapid  stretching  of  the 
muscles  involved  in  the  contraction. 

Try  to  obtain  tendon  jerks  from  other  muscles  ; 
for  example,  the  triceps  humeri,  flexors  of  hand, 
and  masseter  muscles. 

Normally  no  response  will  be  obtained. 

The  experiments  are  of  value  in  diagnosis  of 
diseases  of  the  central  nervous  system. 


8  the  central  nervous  system 

Effect  of  Strychnine  on  Reflex  Action 

Inject  with  a  glass  pipette  a- few  drops  of  0.5 
per  cent  solution  of  sulphate  of  strychnine  into 
the  dorsal  lymph  sac  of  a  frog  the  brain  of  which 
has  been  destroyed  with  a  seeker. 

After  a  few  minutes,  very  weak  afferent 
impulses  will  be  sufficient  to  call  forth  general 
spasmodic  reflex  actions.  Note  that  (1)  the 
strychnine  reflexes  are  paroxysmal,  (2)  the  mus- 
cles fall  into  more  or  less  prolonged  rigidity  (teta- 
nus), and  (3)  the  extensors  overcome  the  flexors, 
the  limbs  being  strongly  extended. 

The  characteristic  action  of  strychnine  is  evi- 
dently not  dependent  on  the  brain. 

Destroy  the  spinal  cord  with  a  seeker. 

Stimulation  of  muscles  and  nerves  will  not 
cause  spasmodic  contractions. 

Strychnine  acts  on  the  spinal  cord,  but  not  on 
the  muscles  or  the  peripheral  nerves. 

Complex  Co-ordinated  Eeflexes 

Removal  of  Cerebral  Hemispheres.  —  Place  a 
frog  under  a  glass  jar  containing  a  small  sponge 
wet  with  ether.  Be  very  careful  not  to  kill  the 
frog.  When  insensibility  is  complete,  place  the 
animal  on  a  frog-board.  Cut  through  the  skin  in 
the  median  line  of  the  skull,  from  the  nose  to  the 


COMPLEX   CO-ORDINATED   REFLEXES  9 

vertebral  column.  Connect  the  front  margins  of 
the  two  tympanic  membranes  by  a  transverse  in- 
cision through  the  skin.  This  transverse  line 
will  pass  over  the  junction  of  the  cerebral  lobes 
with  the  optic  lobes.  Strip  off  the  parietal  bones 
with  forceps,  beginning  at  the  anterior  end  oppo- 
site the  anterior  margin  of  the  orbit.  When  the 
cerebral  hemispheres  are  uncovered,  they  may  be 
removed  from  before  backwards.  Avoid  injuring 
the  optic  lobes.  Work  rapidly  but  carefully.  If 
the  ether  effect  diminish  before  the  operation  be 
finished,  replace  the  frog  under  the  glass  jar  for 
a  few  moments.  As  soon  as  the  hemispheres  are- 
removed,  sew  up  the  wounds  in  the  skin. 

Note  the  signs  of  profound  inhibition. 

If  the  operation  be  done  carefully,  the  shock 
will  gradually  pass  away,  and  the  functions  possi- 
ble in  the  absence  of  the  cerebrum  may  then  be 
determined.  Put  the  frog  aside,  moistening  his 
skin  occasionally,  but  not  otherwise  disturbing 
him,  and  prepare  a  second  frog  for  the  experi- 
ment upon  the  "croak  reflex"  (page  10).  When 
this  operation  is  completed,  resume  the  observa- 
tions on  the  first  frog,  while  the  second  frog  re- 
covers from  the  shock. 

1.  Posture,  etc.  — Write  down  the  differences 
between  the  frog  from  which  only  the  cerebral 
hemispheres  have  been   removed  and  a   frog  in 


10       THE  CENTRAL  NERVOUS  SYSTEM 

which  the  whole  brain  has  been  destroyed  with 
the  seeker,  in  respect  to  posture,  power  to  regain 
feet  when  laid  on  back,  respiratory  movements, 
position  of  eyelids,  leaping  and   swimming. 

2.  Balancing  Experiment.  —  Place  the  frog  on  a 
somewhat  roughened  board,  about  20  inches  long, 
8  inches  wide,  and  1  inch  thick.  Tilt  the 
board  gradually. 

The  frog  remains  motionless  until  his  centre 
of  gravity  is  disturbed.  He  then  moves  forward 
in  an  attempt  to  reach  a  stable  position.  By 
careful  management,  he  can  be  made  to  climb  up 
the  inclined  board,  perch  upon  the  narrow  edge, 
and,  the  board  still  turning,  descend  head-first  on 
the  opposite  side. 

3.  Retinal  Reflex.  —  Place  the  frog  deprived  of 
cerebral  hemispheres  in  front  of  a  bright  light; 
for  example,  an  incandescent  electric  lamp.  In- 
terpose some  object,  such  as  a  small  instrument 
case,  between  the  light  and  the  frog,  so  that  a 
strong  shadow  is  cast  upon  the  frog's  eyes. 
Stimulate  the  frog  by  pinching  the  skin  of  the 
back. 

The  frog  will  jump,  but  will  avoid  the  object 
which  casts  the  shadow. 

4.  Croak  Reflex.  —  Sever  the  large  hemispheres 
from  the  remainder  of  the  brain  of  another  froif 

o 

by  passing  a  knife  through   the  cranium  to  the 


COMPLEX    CO-ORDINATED   REFLEXES  11 

base  of  the  skull  from  side  to  side  in  a  line  join- 
ing the  anterior  margins  of  the  tympanic  mem- 
brane?. (Where  possible,  a  male  frog  should  be 
selected  for  this  experiment.  Males  may  be  rec- 
ognized by  the  cushion-like  thickening  on  the 
innermost  digit  of  the  manus,  or  hand ;  the  male 
Eana  esculenta  possesses  bladder-like,  resonating 
pouches  connected  on  each  side  with  the  mouth 
cavity.)  After  the  immediate  shock  of  the  opera- 
tion has  passed,  stroke  the  back  over  the  anterior 
half  of  the  spinal  cord. 

Beflex  croaking  will  be  observed. 

The  croak  reflex  can  be  inhibited  by  simultane- 
ous pinching  of  one  of  the  limbs  or  other  strong 
stimulation.     (Compare*page  15.) 

If  the  experiments  on  the  frog  in  which  the 
cerebral  hemispheres  were  extirpated  were  not 
satisfactory,  repeat  them  on  this  frog  in  which 
the  hemispheres  were  simply  separated  from  the 
remainder  of  the  brain. 

These  observations  teach  that  very  complicated 
co-ordinated  actions  are  possible  in  the  absence 
of  the  large  hemispheres.  Only  simple  reflexes 
are  possible  when  the  whole  brain  is  removed. 
Consequently,  the  seat  of  these  complicated  re- 
flexes mast  lie  in  the  brain  between  the  cord  and 
the  cerebral  hemispheres. 


12  the  central  nervous  system 

Apparent  Purpose  in  Reflex  Action 

1.  Destroy  the  brain  of  a  frog  with  the  seeker. 
Dip  small  pieces  of  filter  paper  in  strong  acetic 
acid.  Remove  the  superfluous  acid,  lay  the 
paper  bearing  the  acid  on  (1)  the  frog's  thigh, 
(2)  the  foot,  (3)  the  back.  After  each  stimulation 
note  the  character  of  the  reflex  movement,  and 
then  carefully  wash  the  acid  from  the  skin. 

The  movements  are  related  to  the  areas  stimu- 
lated in  a  certain  purposeful  way.  Efforts  are 
made  apparently  to  brush  away  the  acid  paper. 

2.  Place  the  acid  on  the  flank  of  the  right  leg. 
Usually  the  leg  stimulated  strives  to  brush  away 
the  paper.     Hold  this  leg  fast. 

The  other  leg  (the  left)  will  be  used  to  re- 
move the  acid  from  the  opposite  limb.  (This 
experiment  succeeds  best  in  strong,  lively  frogs.) 

3.  Place  an  uninjured  frog  in  an  evaporating 
basin  containing  sufficient  water  to  immerse  the 
frog  to  the  neck  and  covered  with  wire  gauze 
to  keep  him  from  jumping  out.  Warm  the 
water. 

As  the  temperature  rises  to  from  20°-30°  C. 
the  frog  will  attempt  to  escape. 

Repeat  the  experiment  with  the  frog  the  brain 
of  which  has  been  destroyed. 

No    movements    of    escane    will    be    noticed. 


KEFLEX  AND  REACTION  TIME        13 

About  35°,  muscular  twitchings  will  be  seen. 
At  38°-40°  death  takes  place  and  the  muscles 
become  rigid  (heat  rigor). 

This  observation  shows  that  volition  in  all 
probability  is  absent  in  the  brainless  frog.  It 
follows  that  reflex  actions  are  not  volitional; 
their  "  purpose  "  is  only  apparent. 

Keflex  and  Eeaction  Time 

Reflex  Time.  —  Destroy  the  brain  of  a  frog  with 
the  seeker.  Hold  one  leg  of  the  frog  aside  with 
the  glass  rod.  Bring  beneath  the  other  a  small 
beaker  almost  full  of  dilute  sulphuric  acid 
(2:1000).  Raise  the  beaker  until  the  foot  is 
immersed  to  the  ankle.*  Count  the  seconds  be- 
tween the  application  of  the  stimulus  (sulphuric 
acid)  and  the-  withdrawal  of  the  foot. 

This  interval  is  the  reflex  time. 
■  Wash  the  foot  carefully  in  the  bowl  of  water. 

Reaction  Time.  —  Smoke  a  drum.  Raise  the 
drum  off  its  friction  bearing  by  turning  the  screw 
at  the  top  of  the,  shaft.  Place  the  writing  point 
of  an  electromagnetic  signal  against  the  smoked 
paper.  Arrange  a  tuning  fork  to  write  its  curve 
near  that  of  the  signal.  Connect  the  signal 
through  two  simple  keys  and  a  dry  cell  with  the 
primary  coil  of  an  inductorium  arranged  for 
maximal  single  induction  currents  (posts  1  and 


14       THE  CENTRAL  NERVOUS  SYSTEM 

2).  Let  stimulating  electrodes  pass  from  the 
secondary  coil  (bridge  up)  to  the  tongue  of  the 
subject.  Let  the  subject  hold  one  key  closed  un- 
til he  feels  the  stimulus  on  the  tongue. 

Direct  the  subject  to  shut  his  eyes.  Let  the  ob- 
server start  the  tuning  fork,  spin  the  drum,  and 
stimulate  the  subject  by  completing  the  primary 
circuit.  The  instant  the  subject  perceives  the 
stimulus,  he  will  break  the  circuit  by  releasing 
his  key.  By  means  of  the  tuning  fork  curve 
determine  the  interval  between  stimulation  and 
response.  This  interval  is  the  reaction  time  plus 
the  errors  of  observation  ;  for  example,  the  latent 
period  of  the  electromagnetic  signal.  Eepeat  the 
experiment  three  times  and  take  the  mean  of  the 
results. 

In  the  laboratory  note-book  make  a  list  of  the 
links  in  the  chain  between  stimulus  and  re- 
sponse, and  state  as  far  as  possible  the  errors  of 
observation. 

Reaction  Time  with  Choice.  —  Connect  the 
side  cups  of  a  pole-changer  (without  cross  wires) 
to  the  posts  of  the  secondary  coil.  Connect  one 
pair  of  end  cups  with  the  usual  stimulating  elec- 
trodes, the  other  pair  with  large  brass  electrodes 
covered  with  wet  cotton.  Let  the  ordinary  elec- 
trodes touch  the  forehead,  the  other  pair  the  hand 
of  the  subject.     The  other  connections  should  re- 


INHIBITION    OF   REFLEXES  15 

main  as  before.  Eepeat  the  preceding  experi- 
ment but  tell  the  subject  to  signal  only  when 
the  tongue  (or  hand)  is  stimulated.  In  order  to 
do  this  he  must  add  to  his  former  reaction  a  de- 
cision as  to  the  part  stimulated. 

Eeaction  time  with  choice  is  longer  than  sim- 
ple reaction  time.  In  general,  the  more  compli- 
cated the  mental  processes  involved,  the  longer 
will  be  the  reaction  time. 

Inhibition  of  Eeflexes 

Through  Peripheral  Afferent  Nerves.  —  Expose 
the  left  sciatic  nerve  for  a  distance  of  about  15 
mm.  in  a  frog  the  brain  of  which  has  been  de- 
stroyed. Tie  a  thread  around  the  distal  end,  and 
sever  the  nerve  at  the  peripheral  side  of  the  liga- 
ture. Place  the  central  stump  of  the  nerve  on 
the  electrodes  of  the  inductorium,  the  short-cir- 
cuiting key  being  closed.  Make  the  primary 
circuit,  and  set  the  hammer  vibrating.  Xow  open 
the  short-circuiting  key,,  bring  the  right  foot  of 
the  frog  into  the  dilute  sulphuric  acid  up  to  the 
ankle,  and  count  the  seconds  from  the  moment 
of  immersion  to  the  moment  of  withdrawal,  con- 
tinuing meanwhile  the  stimulation  of  the  central 
end  of  the  left  sciatic  nerve. 

The  latent  period  will  be  much  prolonged. 

Wash  off  the  acid  carefully. 


16  THE   CENTRAL    NERVOUS    SYSTEM 

Keflex  actions  may  be  inhibited  by  me  simul- 
taneous stimulation  of  sensory  nerves. 

Through  Central  Afferent  Paths ;  the  Optic 
Lobes.  —  1.  Expose  the  brain  according  to  the 
directions  already  given  (page  8).  Immediately 
posterior  to  the  cerebral  hemispheres  lie  the  optic 
lobes,  two  gray  spherical  bodies.  Separate  the 
cerebral  hemispheres  from  the  optic  lobes  by  a 
transverse  incision,  and  carefully  remove  the 
hemispheres.  Wait  until  the  shock  of  the  opera- 
tion has  passed.  Now  suspend  the  frog  so  that 
the  tips  of  the  toes  hang  above  a  shallow  dish 
containing  water  made  stron^lv  sour  to  the  taste 
with  dilute  sulphuric  acid.  Determine  the  reflex 
time.  \Vash  off  the  acid  and,  after  a  moment's 
rest,  sprinkle  a  very  little  finely  powdered  com- 
mon salt  on  the  cut  surface  of  the  optic  lobes. 
Again  determine  the  reflex  time. 

The  reflex  time  will  be  found  to  be  markedly 
increased  by  the  stimulation  of  the  optic  lobes. 

2.  Prepare  a  second  frog  in  the  same  manner. 
Determine  the  reflex  time.  Now  instead  of  stim- 
ulating the  optic  lobes,  remove  them,  and  again 
determine  the  reflex  time. 

The  removal  of  the  optic  lobes  shortens  the 
reflex  time. 


the  roots  of  spinal  nerves  17 

The  Eoots  of  Spinal  Nerves 
Destroy  the  brain  of  a  strong,  large  frog  with 
a  seeker.  Divide  the  skin  over  the  vertebral 
column  from  the  upper  end  of  the  urostyle  to 
the  level  of  the  fore  limbs.  Hook  back  the 
flaps  of  skin.  Remove  the  longitudinal  muscles 
on  either  side  of  the  spines  of  the  vertebrae,  thus 
exposing  the  bony  arches.  Saw  through  the 
arches  of  the  8th,  7th,  and  6th  vertebra?  (there 
are  ten  vertebrae  in  the  frog,  counting  the  uro- 
style) in  the  order  named.  Clear  away  the  bone, 
and  the  underlying  tissues  until  the  last  three  or 
four  pairs  of  roots  shall  be  plainly  seen.  Grasp 
the  filum  terminale  and  cautiously  lift  the  cord 
until  the  spinal  nerve  roots  are  clearly  displayed. 
The  anterior  roots  are  hidden  by  the  large, 
superficial  posterior  roots.  The  conspicuous  pos- 
terior root  which  seems  to  be  the  last  is,  in  real- 
ity, the  9th,  the  next  to  the  last ;  the  last,  or 
10th,  is  smaller  and  lies  close  to  the  filum  termi- 
nale. Place  a  silk  ligature  about  the  middle 
of  an  anterior  and  a  posterior  root  on  the  right 
side.  With  single  induction  currents  as  stimuli 
observe  that  (1)  the  stimulation  of  only  the  cen- 
tral end  of  the  posterior  root  calls  forth  a  (re- 
flex) movement,  and  (2)  the  stimulation  of  only 
the  peripheral  segment  of  the  anterior  root  causes 
movement.  o 


18       THE  CENTRAL  -NERVOUS  SYSTEM 

On  this  same  side  cut  all  the  posterior  roots. 

No  stimulus  applied  to  the  right  leg  will  now 
discharge  a  reflex  action.  But  stimuli  applied  to 
sensory  nerves  elsewhere  may  still  cause  reflex 
movements  of  the  right  leg.  Motor  impulses  still 
pass  to  these  muscles.  But  only  the  interior 
roots  remain.   . 

Hence  the  anterior  roots  of  spinal  nerves  trans- 
mit motor  impulses  from  the  spinal  cord  towards 
the  muscles  (efferent  impulses) ;  the  posterior 
roots  transmit  sensory  impulses  from  sensory  sur- 
faces towards  the  spinal  cord  (afferent  impulses.) 

Imdwig's  Demonstration.  — Destroy  the  brain 
of  a  large  frog  with  the  seeker.  Kemove  the 
thoracic  and  abdominal  viscera,  taking  care  not 
to  injure  the  sciatic  nerve  plexus.  Remove  the 
7th  and  8th  vertebrae,  taking  the  greatest  pains 
not  to  injure  the  nerve  roots.  Divide  the  body 
transversely  at  this  level,  so  that  the  anterior 
and  posterior  halves  shall  remain  connected  only 
by  the  anterior  and  posterior  sciatic  roots.  Keep 
the   roots    moist    with    normal    saline    solution. 

Demonstrate  again  that  the  anterior  roots 
transmit  efferent,  and  the  posterior  roots  afferent 
impulses. 

Localization  of  Movements  at  Different  Levels  of 
the  Spinal  Cord.  —  Separate  the  three  roots  which 
form   the  sciatic   nerve.     After  tying  a  thread 


DISTRIBUTION   OF   SENSORY   SPINAL   NERVES      19 

about  each  root  sever  it  from  the  spinal  cord  by 
a  cut  on  the  proximal  side  of  the  thread.  Stimu- 
late each  nerve  with  a  very  weak  tetanizing  cur- 
rent. Note  the  different  results,  obtained  from 
nerves  arising  at  different  levels  of  the  cord. 
Stimulation  of  the  most  anterior  root  causes 
marked  flexion  of  the  limb ;  stimulation  of  the 
middle  roots,  extension  and  internal  rotation ; 
and  of  the  most  posterior,  simple  extension. 

In  a  frog  whose  nerves  have  not  been  cut 
expose  the  spinal  cord  and  stimulate  it  at  differ- 
ent levels  in  both  directions  along  its  length. 
The  various  movements  of  the  hind  limbs  are 
localized  at  different  levels  of  the  cord. 

Distribution  of  Sensory  Spinal  Nerves 

Destroy  the  brain  of  a  large  frog  with  the 
seeker.  Expose  the  lower  half  of  the  spinal  cord 
by  the  method  already  described.  On  one  side 
cut  the  dorsal  sensory  root  of  the  8th  spinal  nerve 
and  on  the  other  cut  the  sensory  root  of  the  7th, 
9th,  and  10th.  After  the  section  of  each  root 
test  the  cutaneous  sensibility  of  the  limbs  by 
placing  upon  the  skin  small  pieces  of  filter  paper 
(two  mm.  square)  moistened,  not  dripping,  with 
0.2  per  cent  sulphuric  acid.  Make  a  map  of  the 
anaesthetic  areas  in  each  leg,  and  note  the  lack 
of  correspondence. 


20       THE  CENTRAL  NERVOUS  SYSTEM 

Many  skin  areas  are  supplied  by  fibres  from  at 
least  two  sensory  roots.  The  fields  of  distribution 
overlap. 

Muscular  Tonus 

Brondgeest's  Experiment.  —  Fasten  a  lightly 
etherized  frog  back  uppermost  on  the  frog-board. 

In  a  line  between  the  ilium  and  the  coccyx 
open  the  pelvic  cavity  by  cautiously  dividing  the 
skin,  fascia,  and  muscle.  Divide  the  sciatic  nerve 
roots  on  the  operated  side.  Pass  a  hook  or  thread 
through  the  jaw  and  hang  the  frog  up. 

Observe  that  the  limb  the  nerves  of  which 
have  been  cut  is  relaxed,  so  that  the  toes  hang 
lower  than  those  of  the  limb  which  still  retains 
its  connection  with  the  central  nervous  system. 

Apparatus 

Normal  saline.  Bowl.  Towel.  Pipette.  Stand. 
Muscle  clamp.  Bent  hook.  Inductorium.  Dry  cell. 
Electrodes.  Large  brass  electrodes.  Cotton.  Key. 
Frog-board.  Fine  copper  wire.  One-half  per  cent  solu- 
tion of  strychnine  sulphate.  Glass  jar  with  ether  and 
sponge.  Balancing  board.  Strong  acetic  acid.  Filter 
paper.  Evaporating  basin.  Wire  gauze.  Bunsen  burner. 
Thermometer.  Dilute  sulphuric  acid  (0.2  per  cent). 
Beaker.  Kymograph.  Electromagnetic  signal.  Tuning 
fork.     Pole-changer.     Vertebral  saw. 


II 

THE   SKIN 
Sensations  of  Temperature 

Hot  and  Cold  Spots. — With  a  lead-pencil  point 
carefully  explore  an  area  about  an  inch  square 
on  the  back  of  the  wrist  or  hand.  Mark  with 
black  ink  the  places  where  a  distinct  sensation  of 
cold  is  felt,  and  with  red  ink  those  where  the 
-sensation  is  one  of  warmth. 

The  places  indicated  are  the  so-called  hot  and 
cold  spots. 

Outline.  —  Attempt  to  define  more  exactly  the 
outline  of  one  of  the  cold  spots. 

The  spots  are  of  irregular  shape,  —  blotches 
rather  than  points. 

Mechanical  Stimulation.  —  1.  Gently  tap  one 
end  of  a  small  wooden  rod  the  other  end  of  which 
is  placed  on  a  well-defined  cold  spot. 

The  mechanical  stimulation  of  the  cold  spot 
will  give  a  sensation  of  cold. 

2.    Stimulate  a  warm  spot  mechanically. 

Chemical  Stimulation.  —  Pi  lib  a  menthol  pencil 
over  a  small  area  on  the  back  of  the  hand. 


22  THE   SKIN 

A  sensation  of  cold  will  be  perceived.  This  is 
due  to  chemical  irritation  of  the  cold  spots.  The 
temperature  of  the  area  does  not  fall. 

Electrical  Stimulation.  —  It  has  been  found  that 
the  stimulation  of  a  well-defined  cold  or  warm 
spot  with  moderately  strong  induced  currents 
causes  a  sensation  of  cold  or  warmth  respectively. 

Temperature  After-Sensation. — Stimulate  a  cold 
spot  mechanically  with  a  pencil  point.  Remove 
the  point. 

The  sensation  of  cold  outlasts  the  stimulus. 

Balance  between  Loss  and  Gain  of  Heat.  —  Pro- 
vide three  beakers  of  water.  Heat  them  to  20°, 
30°,  and  40°  C,  respectively.  Place  a  finger  of 
one  hand  in  the  water  at  20°,  and  a  ringer  of  the 
other  hand  in  the  water  at  40°.  After  the  re- 
spective sensations  of  cold  and  warmth  have 
disappeared,  place  both  the  fingers  in  the  water 
at  30°. 

The  finger  from  the  cold  water  will  seem  warm 
and  that  from  the  warm  water  cold.  The  tem- 
perature of  the  skin  equals  the  balance  between 
its  heat  loss  and  heat  gain.  When  this  tempera- 
ture is  raised  or  lowered,  the  warm  spots  or  cold 
spots  respectively  are  stimulated. 

Fatigue.  —  Provide  three  beakers  containing 
water  at  10°,  32°,  and  45°  C.  respectively.  Place 
a  finger  of  one  hand  in  the  beaker  at  32°,  and  a 


SENSATIONS   OF   TEMPERATURE  23 

finder  of  the  other  hand  in  the  beaker  at  45°. 
After  .45  seconds  place  both  fingers  in  the  water 
at  10°. 

The  finger  taken  from  the  water  at  32°  (which 
is  about  the  normal  temperature  of  the  hand)  will 
feel  colder  than  the  other  finger.  Extreme  tem- 
peratures of  heat  or  cold  fatigue  the  temperature 
spots. 

Relation  of  Stimulated  Area  to  Sensation. — In- 
sert a  finger  of  one  hand  in  a  beaker  of  warm  or 
cold  water.  Note  the  sensation.  Insert  a  finger 
of  the  other  hand  in  the  water. 

The  intensity  of  the  sensation  will  increase 
with  the  extent  of  the  surface  stimulated. 

Perception  of  Difference.  — Provide  two  beakers 
of  water, one  at  30°,  the  other  slightly  warmer  or 
colder.  By  introducing  a  finger  first  into  the  one 
and  then  into  the  other,  and  varying  the  tem- 
perature of  the  water,  ascertain  how  small  a 
difference  in  temperature  can  be  detected. 

Usually  a  difference  of  0.5°  C.  is  easily  recog- 
nized. 

Relatively  Insensitive  Regions.  —  1.  Compare 
the  temperature  sensation  perceived  on  touching 
with  a  pencil  point  the  median  line  of  the  fore- 
head, nose,  and  chin  with  that  perceived  on 
touching  the  skin  on  either  side  of  the  median 
line. 


24  THE    SKIN 

The  skin  in  the  median  line  of.  the  body  is 
comparatively  insensitive  to  temperature  varia- 
tions. 

2.  Similarly  compare  the  mucous  membrane 
with  the  skin. 

The  mucous  membranes  are  much  less  sensitive 
than  the  skin. 

Sensations  of  Pressure 

Pressure  Spots.  —  Explore  the  surface  of  the 
forearm  by  bringing  the  blunted  point  of  a  needle 
gently  in  touch  with  the  skin. 

At  certain  spots  a  distinct  sensation  of  contact 
will  be  perceived.  Other  spots  will  give  only 
dull  sensations.  Pressure,  like  heat  and  cold,  is 
appreciated  by  scattered  sense-organs  in  the  skin, 
not  by  diffuse  general  sensation. 
.  Note  the  relation  of  the  pressure  points  (1)  to 
the  hair  follicles,  and  (2)  to  the  warm  and  cold 
spots  mapped  out  in  previous  experiments. 

Threshold  Value.  —  Take  from  the  human  head 
several  straight,  strong  hairs.  Cement  each  to 
the  end  of  a  little  stick  of  soft  pine  to  serve  as 
a  handle.  Provide  a  special  lever,  made  as  fol- 
lows :  With  a  hot  pin  burn  a  small  hole  at  the 
middle  of  a  straw  about  25  cm.  in  length.  Pass  a 
needle  through  this  hole  into  a  cork  held  in  the 
muscle  clamp.     Press  the  free  end  of  the  hairs 


SENSATIONS    OF   PRESSURE  25 

against  different  parts  of  the  skin  of  the  hand. 
arm,  and  face.  Select  hairs  which  when  pressed 
against  the  skin  of  the  respective  regions  give  no 
sensation  of  pressure.  Shorten  the  hairs  until 
the  pressure  is  just  perceptible.  This  will  be  the 
"pressure  threshold."  Make  a  loop  in  a  short 
silk  thread  and  pass  the  loop  about  the  lever 
exactly  one  millimetre  from  the  axis.  Hang  on 
the  end  of  the  thread  a  light  bent  hook.  Coun- 
terpoise  the  lever  very  exactly,  so  that .  the 
slightest  force  applied  to  the  end  of  the  straw 
will  raise  the  lever  from  the  after-loading  screw. 
By  counterpoising  in  this  way,  the  lever  becomes 
a  balance.  On  the  bent  hook  hang  a  ring  of 
German  silver  wire  weighing  one  decigram  (0.1 
gram).  Find  a  point  on  the  lever  100  mm.  from 
the  axis.  The  weight  of  one  decigram  suspended 
1  mm.  from  the  axis  of  the  lever  will  be  raised 
by  a  force  of  t^q  of  a  decigram,  equal  to  one 
milligram  (0.001  gram)  applied  100  mm.  from 
the  axis.  At  50  mm.  from  the  axis,  0.1  gram, 
suspended  1  mm.  from  the  axis,  will  be  lifted  by 
a  force  of  ^Jn  gram  (0.002  gram).  Find  the  dis- 
tance from  the  axis  at  which  each  testing-hair, 
when  pressed  vertically  against  the  lever,  will 
just  fail  to  lift  the  lever;  in  other  words,  the 
point  at  which  the  pressure  will  be  just  sufficient 
to  bend  the  hair.      The  number  of  millimetres 


26  THE    SKIN 

between  this  point  and  the  axis  of  the  lever, 
multiplied  by  one-tenth,  will  give  the  bending 
pressure  of  the  hair  in  the  fraction  of  a  grain. 
Make  ten  observations  on  each  hair  and  mark  the 
mean  bending  value  on  the  wooden  handle. 

Touch  Discrimination.  —  1.  Close  the  eyes  and 
let  an  assistant  test  the  different  parts  of  the 
skin  of  the  hand,  arm,  and  face  for  discrimina- 
ting power.  For  each  test  separate  the  points  of 
the  aesthesiometer  until  they  can  be  felt  as  two 
(ordinary  drawing  dividers  or  compasses  can  be 
used  for  an  aesthesiometer). 

Eecord  your  results  in  millimetres  for  finger- 
tips, palm  of  hand,  back  of  fingers,  back  of  hand, 
back  of  wrist,  flexor  and  extensor  surfaces  of  fore- 
arm, forehead,  cheeks,  lips,  and  tongue. 

2.  Separate  the  points  of  the  aesthesiometer 
about  20  mm.,  and  draw  them  gently  side  by 
side  along  the  extensor  surface  of  the  forearm 
from  the  elbow  to  the  wrist.  Eepeat  the  experi- 
ment on  the  flexor  surface.  Try  the  same  for 
the  cheek  and  lips,  beginning  near  the  ear  and 
drawing  the  points  so  that  one  shall  go  above 
and  the  other  below  the  mouth. 

Describe  the  sensation  in  each  case,  and  sug- 
gest an  explanation. 

Weber's  Law.  —  Place  the  hand  palm  upward 
in  a  comfortable  position   on   the  table.     Close 


SENSATIONS    OF   PRESSURE  27 

the  eyes.  Let  an  assistant  place  on  the  last 
phalanx  of  the  middle  and  index  fingers  a  small 
round  box  containing  ten  small  shot. 

When  the  subject  has  formed  a  clear  percep- 
tion of  the  weight,  let  an  assistant  add  or 
subtract  shot,  and  record  the  number  of  shot  cor- 
responding .  to  the  smallest  difference  in  weight 
perceived  by  the  subject  (whose  eyes  of  course 
should  be  kept  closed).  Repeat  the  experiment 
with  20,  30,  40,  and  50  shot  in  the  box  respec- 
tively. Determine  in  each  instance  the  ratio  of 
the  number  of  shot  added  or  subtracted  to  the 
number  with  which  each  experiment  was  begun. 

This  ratio  will  be  approximately  constant. 
The  degree  of  stimulation^necessary  to  cause  the 
perception  of  difference  always  bears  the  same 
ratio  to  the  degree  of  stimulation  already  applied. 
Weber's  law  is  less  true  for  very  small  and  very 
large  weights  than  for  those  of  medium  value. 
It  is  a  general  law  and  holds  good  for  visual 
judgments,  etc. 

After-Sensation  of  Pressure.  —  Place  a  rubber 
band  about  the  head  and  allow  it  to  remain  for 
several  minutes. 

On  removing  the  band,  a  distinct  after-sensa- 
tion of  pressure  will  be  felt. 

Temperature  and  Pressure.  — Place  on  the  back 
of  the  hand  supported  on  the  table  a  coin  the 


28  THE   SKIN" 

temperature  of  which  has  been  made  such  that 
it  feels  neither  warm  nor  cold.  Compare  the 
pressure  sensation  (apparent  weight)  of  this  "nor- 
mal" coin  with  that  of  similar  coins  warmed 
and  cooled. 

The  hot  or  cold  coin  will  seem  heavier  than 
the  "  normal "  coin  of  equal  weight. 

Touch  Illusion  ;  Aristotle's  Experiment.  —  Cross 
the  right  middle  ringer  over  the  right  index  finger 
and  place  them  on  the  palm  of  the  left  hand. 
Place  a  small  shot  between  the  crossed  ringers  in 
such  a  way  that  it  shall  touch  the  ulnar  side  of 
the  middle  finger  and  the  radial  side  of  the  index 
finger.     Eoll  the  shot  in  the  palm  of  the  hand. 

A  sensation  of  two  objects  will  be  felt. 

Apparatus 

Black  and  red  ink.  Small  wooden  rod.  Menthol  pen- 
cil. Inductorium.  Dry  cell.  Electrodes.  Key.  Three 
beakers.  Stand.  Ring.  Wire  gauze.  Bunsen  burner. 
Thermometer.  Needle  with  blunted  point.  Muscle  lever. 
Gram  and  ten-gram  weights.  German  silver  ring  weigh- 
ing 0.1  gram.  Silk  thread.  Four  small  wooden  handles 
for  pressure-hairs.  Bent  hook.  Drawing  dividers  (as 
sesthesiometer).  Small  round  box  containing  at  least  50 
shot.     Rubber  band  larce  enough  to  go  around  the  head. 


TICKLE  29 

III* 

GENERAL   SENSATIONS 

Tickle 

Irradiation.  —  Gently  touch  the  skin  near  one 
nostril  with  a  dry  camel' s-hair  brush. 

Note  (1)  the  strong  sensation  produced  by 
the  slight  stimulus ;  (2)  the  irradiation  beyond 
the  spot  stimulated. 

After  image.  —  Repeat  the  stimulus  of  the  pre- 
ceding experiment.  ■ 

Measure  in  seconds  the  time  during  which 
the  sensation  outlasts  the  stimulus  (after  image). 

Topography.  — Test  the  tickle  sensation  at  vari- 
ous points  on  the  skin  etf  the  face,  hands,  and 
forearms.  Determine  whether  the  sensation  is 
greatest  about  the  several  openings,  where  skin 
joins  mucous  or  serous  membranes ;  e.  g.,  the 
nostrils,  the  conjunctival  sac,  the  auditory  canal. 
Do  the  results  indicate  a  protective  mechanism  ? 

Summation.  —  In  one  of  the  sensitive  areas 
found  in  the  preceding  experiment  determine  the 
difference  between  the  response  to  a  single  stim- 
ulus and  to  successive  stimuli. 

Fatigue.  —  In  any  sensitive  area  determine  (1) 
the  quickness  with  which  the  apparatus  for  the 
sensation  of  tickle  is  fatigued ;  (2)  the  duration 
of  fatigue. 


30  GENERAL   SENSATIONS 

Pain 

Threshold  Value.  —  Arrange  an  inductorium  for 
tetanizin"  currents.  Place  the  electrodes  on 
the  tip  of  the  tongue,  and  move  the  secondary 
toward  the  primary  coil  until  no  farther  move- 
ment can  be  made  without  causing  the  stimula- 
tion to  become  painful.  Determine  for  this 
region  and  for  others  of  the  mucous  membrane 
of  the  mouth  and  of  the  skin  what  distance  of 
the  secondary  coil  from  the  primary  separates  the 
stimulus  at  which  pain  is  just  perceived  from 
that  at  which  the  pain  is  distinct. 

Latent  Period.  —  In  several  individuals  measure 
approximately  the  interval  between  the  applica- 
tion of  the  stimulus  (single  break  shock)  and  the 
resulting  painful  sensation. 

Summation. — Determine  the  number  of  sub- 
minimal stimuli  necessary  to  produce  pain. 

Topography.  —  Map  upon  the  skin  of  the  face 
and  arm  the  areas  specially  sensitive  to  pain. 

Individual  Variation.  —  Compare  the  reactions 
of  several  individuals,  and  note  the  differences  in 
threshold  value,  latent  period,  summation,  and 
topography. 

Temperature  Stimuli.  —  Fill  two  bowls  or  large 
beakers  with  water  twenty-five  degrees  respec- 
tively, hotter  and  colder,  than  the  temperature 


MOTOR    SENSATIONS  31 

of  the  hand.  Determine  whether  the  increase 
or  the  corresponding  decrease  in  temperature  is 
the  more  painful  to  the  immersed  hand. 

Motor  Sensations 

Judgment  of  Weight.  —  Lift  the  same  weight 
twice,  at  first  very  slowly  and  then  quickly. 

The  weight  will  appear  lighter  when  raised 
quickly. 

Sensation  of  Effort.  —  "  Hold  the  finger  as  if  to 
pull  the  trigger  of  a  pistol.  Think  vigorously 
of  bending  the  finger,  but  do  not  bend  it. 

"  An  unmistakable  feeling  of  effort  results. 

"  Repeat  the  experiment,  and  notice  that  the 
breath  is  involuntarily  held,  and  that  there  are 
tensions  in  other  muscles  than  those  that  would 
move  the  finger."     (Sanford.) 

Sensation  of  Motion.  —  Let  the  forearm  and 
hand  rest  upon  a  table.  Bring  the  four  fingers 
of  the  hand  together,  and  turn  the  hand  so  that 
it  shall  rest  upright  upon  the  ulnar  side  of  the 
little  finger.  Close  the  eyes.  Abduct  the  first 
finger. 

The  second,  third,  and  fourth  finger  will  seem 
to  move  in  a  direction  opposite  to  the  movement 
of  the  first. 


32  TASTE 

IV 
TASTE 

Threshold  Value.  —  Prepare  solutions  of  cane 
sugar  of  the  following  strengths :  1  :  1000, 1  :  800, 
1  :  600,  1  :  400,  1  :  200,  1  :  100.  Take  half  a 
tea'spoonful  of  the  weakest  solution  into  the 
mouth,  roll  it  upon  the  tongue,  and  swallow 
it.  Note  whether  a  sweet  taste  can  be  per- 
ceived. Rinse  the  mouth  thoroughly.  Proceed 
with  solutions  of  increasing  strength  until  the 
sweet  taste  is  just  perceptible. 

Topography.  —  1.  Select  a  solution  of  sugar 
slightly  more  concentrated  than  that  just  per- 
ceived to  be  sweet.  With  a  small  camel's-hair 
brush  apply  this  solution  to  the  several  parts 
of  the  tongue  and  the  palate.  Determine  the 
regions  sensitive  to  taste.  The  mouth  must  be 
rinsed  frequently.  2.  Dry  the  upper  surface  of 
the  tongue  with  a  handkerchief.  With  a  finely 
pointed  camel's-hair  brush  apply  a  twenty  per 
cent  sugar  solution  to  the  individual  fungiform 
papillae  and  to  the  mucous  membrane  between 
them.  Determine  whether  only  the  papillse 
perceive  taste. 

Relation  of  Taste  to  Area  stimulated.  —  Swallow 
a  very  small  quantity  of  a  minimal  solution  of 
sugar,  as   determined   in   the  experiment    upon 


vision  33 

threshold  value.  Rinse  the  mouth,"  and  then 
swallow  a  much  larger  portion  of  the  solution. 

The  taste  will  be  perceived  more  strongly,  the 
larger  the  area  stimulated. 

Electrical  Stimulation.  — 1.  Connect  two  small 
zinc  electrodes  through  a  simple  key  to  a  battery 
of  four  dry -cells.  Apply  one  electrode  to  an  in- 
different region,  the  other  to  the  tongue.  Close 
the  key. 

Note  the  sour  taste  at  the  positive  pole  and 
the  alkaline  taste  at  the  negative. 


VISION 

Mapping  the  Blind  Spot.  —  Fasten  a  rod  fifteen 
inches  from  the  table.  Beneath  the  rod  place 
a  well-lighted  sheet  of  white  paper  (a  page  of 
the  laboratory  note-book  will  serve).  Make  a 
small  black  cross  near  the  left  margin.  Eest 
the  chin  upon  the  rod  in  such  a  way  that  the 
right  eye  shall  look  directly  down  at  the  cross. 
Place  the  hand  over  the  other  eye.  A  straw 
bearing  a  black  pin-head  will  be  drawn  by  an 
assistant  from  the  cross  along  the  horizontal 
meridian  toward  the  temporal  side  of  the  eye 
under  observation.  The  assistant  will  mark  the 
point  where  the  black  object  ceases  to  be  visible, 
and  the  point  at  which  it  -reappears.     These  are 


34  VISION 

the  boundaries  of  the  blind  spot  of  the  right 
eye  in  the  horizontal  meridian.  Determine  the 
boundaries  in  other  meridians.  Obtain  similarly 
the  outlines  of  the  blind  spot  of  the  left  eye. 

Yellow  Spot.  —  Close  the  eyes  for  half  a 
minute,  and  then  look  at  the  clear  sky  or  a 
brightly  lighted  surface  through  a  solution  of 
chrome  alum  in  a  glass  bottle  with  parallel 
sides.  The  yellow  spot  will  appear  rose-colored 
in  the  blue-green-red  solution.  The  yellow  pig- 
ment absorbs  some  of  the  blue  and  green  rays. 
The  remaining  rays  form  rose  color. 

Field  of  Vision.  —  Fasten  in  a  vertical  position 
a  sheet  of  white  paper  about  50  cm.  high  and 
60  cm.  broad.  (It  may  be  pinned  to  the  wooden 
stand  set  on  edge  upon  the  electrometer  box.) 
About  20  cm.  from  the  left  margin  and  30  cm. 
from  the  lower  margin  of  the  paper  mark  a  small 
cross.  Let  the  subject  rest  his  chin  upon  a  rod 
clamped  to  the  iron  stand  in  such  a  way  that 
the  right  eye  shall  look  directly  at  the  cross. 
Cement  the  squares  of  black,  red,  green,  and 
blue  papers  to  the  ends  of  separate  straws. 
Carry  the  black  square  from  without  inwards 
along  the  horizontal  meridian  intersecting  the 
cross.  Mark  the  point  at  which  the  black 
object  enters  the  field  of  vision.1  This  point  is 
the  temporal  boundary  of  the  visual  field  in  the 


vision  35 

horizontal  meridian.  Determine  in  the  same 
way  the  boundary  on  the  nasal  side.  Kepeat 
for  several  other  meridians.  A  line  joining  the 
points  obtained  will  bound  the  visual  field. 

Determine  the  visual  field  for  red,  green,  and 
blue.  Always  pass  the  test  color  from  without 
inwards.  The  subject  should  be  ignorant  of 
the  color  to  be  used,  and  should  name  the  color 
as  soon  as  it  enters  his  visual  field. 


Color  Blindness 

The  three  lame  skeins  show  the  test  colors. 

1.  Light  Green.  —  Palest-  (lightest)  shade  of 
very  pure  green,  —  neither  yellow-green  nor 
blue-green  to  the  normal  eye.  Light  green  is 
chosen  because,  according  to  the  Young-Helm-, 
holtz  theory,  it  is  the  whitest  of  the  colors  of 
the  spectrum,  and,  consequently,  is  most  easily 
confused  with  gray.  Light  shades  are  employed 
because  it  is  difficult  to  distinguish  between 
strongly  illuminated  shades. 

2.  Par  pie  (Rose). —  A  skein  midway  between 
lightest  and  darkest  purple.  Chosen  because 
purple  combines  two  fundamental  colors  which 
are  normally  never  confounded. 

3.  Ilecl.  —  A  vivid,  slightly  yellowish  red. 
Chosen  because  it  represents  the  color-group  in. 


36  vision 

which  red  (orange)  and  violet  (blue)  are  com- 
bined in  nearly  equal  proportions. 

Me':hod  of  Examination  and  Diagnosis.  —  Place 
the  Berlin  worsteds  on  the  white  cloth  in  which 
they  are  wrapped.  They  should  be  well  mixed, 
and  not  spread  out  too  much.  Lay  a  skein  of 
the  first  test-color  in  a  well-lighted  position  two 
or  three  feet  from  the  group.  Inform  the  person 
examined : 

(1)  That  he  must  not  speak  during  the  test. 

(2)  That  the  skeins  are  not  to  be  fingered  or 
tossed  about.  A  skein  should  be  touched  only 
after  its  selection, 

(3)  That  he  must  endeavor  to  pick  out  skeins 
resembling  the  test  skein,  i.  e.,  a  little  lighter  or 
darker  in  shade ;  the  resemblance  cannot  be  per- 
fect, as  no  two  shades  are  exactly  alike. 

.   Green  Test.  —  The  subject   must  pick   out  all 
the  other  skeins  approximately  the  same  shade. 
The  color-blind  selects  some  shade  of  gray. 

.•Purple  {Rose).  —  The  subject  should  pick  out 
the  skeins  of  the  same  color,  as  before. 

.  (1)  He  who  is  color-blind  by  the  first  test,  and 
who,  upon  the  second  test,  selects  only  purple 
skeins,  is  incompletely  par  pie-blind. 

(2)  He  who,  in  the  second  test,  selects  with 
purple  only  blue  and  violet,  or  one  of  them,  is 
completely  red-blind. 


vision  37 

(3)  He  who,  in  the  second  test,  selects  with 
purple  only  green  and  gray,  or  one  of  them,  is 
completely  green-blind. 

Remark.  —  The  red-blind  never  selects  the 
colors  taken  by  the  green-blind,  and  vice  versa. 
Often  the  green-blind  places  a  violet  or  blue 
skein  by  the  side  of  the  green,  but  only  the 
brightest  shades  of  these  colors.  This  does  not 
influence  the  diagnosis. 

Red.  —  This  test  is  applied  to  those  completely 
color-blind.  Continue  the  test  until  the  person 
examined  has  placed  beside  the  specimen  all  the 
skeins  belonging  to  this  shade,  or  else,  separately, 
one  or  more  "  colors  of  contusion." 

The  red-blind  chooses  (besides  the  red,  green, 
and  brown)  shades  which  to  the  normal  sense 
seem  darker  than  red.  The  green-blind  .selects 
opposite  shades,  which  seem  lighter  than  red. 

Violet  Blindness.  —  Very  rare.  Recognized  by 
a  confusion  of  purple,  red,  and  orange,  in  the 
purple  test  (see  2).  Much  care  is  required  to 
diagnosticate  this  form. 


38  FERMENTATION 

FERMENTATION 

Specific  Action 

Conversion  of  Starch  to  Sugar  by  Germinating 
Barley.  — To  5  grams  crushed,  germinating  barley 
add  10  grams  potato  starch,  and  20  c.c.  of  cold 
water.  Then  add  gradually  70  c.c.  of  hot  water 
with  constant  stirring.  Keep  the  mixture  in  a 
temperature  of  about  60°  C.  for  one  hour. 

The  insoluble  starch  will  be  converted  to  a 
sweet  liquid.1 

Boil  10  c.c.  of  Fehling's  solution,2  dilute  the 
syrup  with  water  and  add  it  drop  by  drop  to  the 
boiling  Fehling's  solution. 

1  Kiichoff :  Schweigger's  Journal  fur  Chemie  und  Physik, 
1815,  xiv,  p.  3S9. 

2  Fehlirvg 's  solution.—  In  a  large  watch  glass  weigh  34.639  gms. 
pure  cupric  sulphate  (clean  crystals).  Dissolve  the  crystals  by 
warming  them  with  about  150  c.c.  water  in  an  evaporating  dish. 
Place  the  solution  in  a  500-c.c.  measuring  flask.  Wash  the 
remnant  from  the  dish  into  the  flask.  Allow  the  liquid  to  cool 
completely.     Add  water  to  the  mark  on  the  neck  of  the  flask. 

Warm  about  173  gms.  potassium  sodium  tartrate  in  a  little 
water  until  dissolved.  Place  the  solution  in  a  500-c.c.  measur- 
ing flask,  add  100  c.c.  sodium  hydroxide,  sp.  gr.  1.34  (about 
31  per  cent),  and,  after  the  mixture  has  completely  cooled,  fill 
the  flask  to  the  mark  on  the  neck. 

In  use,  mix  equal  volumes  of  each  solution  in  a  dry  glass. 

One  molecule  grape  sugar  reduces  five  molecules  cupric  oxide 
to  cuprous  oxide  ;  10  c.c.  of  Fehling's  solution  equals  0.05  gin. 
grape  sugar. 


SPECIFIC   ACTION  6\) 

Red  cuprous  oxide  or  its  yellow  hydrate  will 
separate. 

The  germinating  barlev  causes  the  starch  to 
take  up  water,  thus  changing  to  a  reducing  sugar. 
In  this  instance  the  agent  is  a  living  cell,  or 
some  substance  or  "  ferment "  secreted  by  the 
cell.  It  is  now  necessary  to  inquire  whether 
ferments  are  separable  from  living  cells. 

Conversion  of  Starch  to  Sugar  by  Salivary  Dias- 
tase (Ptyalin).  —  To  10  c.c.  of  starch  paste1  col- 
ored blue  with  iodine  (blue  iodide  of  starch)  add 
about  2  c.c.  of  filtered  saliva  and  keep  the  mixture 
at  35-40°  C. 

The  starch  paste  will  liquefy  and  become  sweet. 
The  blue  color  will  become  lighter  and  finally 
disappear. 

Test  with  Fehling's  solution.  Eeduction  will 
take  place. 

Saliva  hydrolyzes  starch  to  a  reducing  sugar. 

Saliva  is  secreted  by  the  cells  in  the  salivary 
gland,  placed  some  distance  from  the  mouth. 
The  saliva  itself  contains  no  secreting  cells. 
There  are  ferments,  then,  which  act  at  a  distance 

1  Starch  paste.  — Rub  1  gin.  potato  starch  in  a  mortar  with 
25  c.c.  cold  water.  Pour  the  mixture  into  an  evaporating  dish. 
Wash  the  remnant  from  the  mortar  and  pestle  into  the  dish  with 
75  c.c.  water.  Heat  the  mixture  to  boiling  point  with  constant 
stirring.  The  starch  paste  will  turn  blue  upon  addition  of  iodine 
(iodide  of  starch). 


40  FERMENTATION 

from  the  cells  that  produce  them.  There  seems 
thus  an  important  distinction  to  be  made  between 
organized  ferments,  those  acting  apparently  with- 
in the  living  cell,  and  unorganized  ferments,  like 
the  salivary  diastase,  which  is  secreted  by  a 
living  cell  but  remains  active  after  leaving  the 
cell.  It  will  be  seen  that  this  distinction  cannot 
be  maintained. 

Extraction  of  Diastase  from  Germinating  Barley.1 
—  Crush  freshly  germinating  barley  in  a  mortar 
with  about  half  its  weight  of  water.  Keep  the 
mass  two  hours  at  35-40°  C.  Squeeze  out  the 
watery  extract  in  a  press,  or  strain  by  strong 
pressure  through  a  linen  cloth.  Add  excess  of 
alcohol. 

Diastase  will  be  precipitated.  It  may  be  puri- 
fied by  dissolving  it  in  water  and  reprecipitating 
with  alcohol. 

Add  a  little  diastase  to  10  c.c.  starch  paste, 
colored  blue  with  iodine.  The  starch  will  be  con- 
verted to  sugar.     The  blue  color  wTill  disappear. 

It  appears,  therefore,  that  ferment  action  is 
not  dependent  on  the  life  of  the  cell  that  secretes 
the  ferment. 

Specific  Action  of  Ferments.  —  The  question 
now  arises    whether   the   diastase   acts   only  to 

1  Payen  and  Persoz :  Armales  de  chimie  et  de  physique, 
1833,  liii,  p.  78. 


PROTEID   DIGESTION    BY   PEPSIN  41 

change  starch  to  sugar  or  whether  it  causes  the 
decomposition  of  other  substances. 

Place  a  small  piece  of  fibrin  in  a  test-tube  and 
add  2  c.c.  filtered  saliva.  Keep  the  tube  several 
hours  at  a  temperature  of  35-40°  C 

The  fibrin  will  not  change. 

Place  0.5  c.c.  neutral  olive  oil  (page  56)  and  2 
c.c.  filtered  saliva  in  a  test-tube. 

Noteworthy  changes  will  be  absent. 

From  these  experiments  it  is  evident  that 
diastase  decomposes  starches,  but  does  not  de- 
compose proteids  and  fats.  Its  ferment  action  is 
thus  far  "  specific."  The  belief  that  each  ferment 
has  its  own  characteristic  product  will  be  in- 
creased by  the  study  of  the  following  typical 
ferment  actions. 

Peoteid  Digestion  by  Pepsin 

Gastric  Digestion  of  Cooked  Beef  and  Bread.  — 

At  7  a.m.  feed  cooked  beef  and  bread  to  a  cat 
which  has  fasted  twelve  hours.  At  11  A.  M.  kill 
the  cat,  expose  the  stomach,  and  apply  double 
ligatures  about  1  cm.  apart  to  the  duodenum  at 
the  pylorus  and  to  the  oesophagus  at  the  cardiac 
orifice.  Eemove  the  stomach.  Open  the  stomach 
very  cautiously  by  drawing  a  knife  along  the 
greater  curvature. 

"  The  stomach  is  very  full,  and  still  contains 


42  FERMENTATION 

much  meat  and  bread  not  wholly  softened.  The 
softening  is  greater  in  the  portal  region  and  in 
those  portions  of  the  food  next  the  mucous  mem- 
brane than  in  the  middle  of  the  stomach  contents. 
The  mucus  secreted  by  the  gastric  mucous 
membrane-is  very  abundant  and  is  strongly  acid. 
The  stomach  contents  have  a  sour  odor."  1 

Artificial  Gastric  Juice.2  —  1.  Strip  the  milCOUS 
membrane  from  the  fourth  stomach  of  a  calf. 
Wash  the  membrane  with  cold  water  until  the 
acid  reaction  disappears.  Dry  the  mucous  mem- 
brane in  the  air.  Divide  some  of  the  dried 
membrane  into  small  pieces  and  add  dilute  hy- 
drochloric acid.3 

2.  Strip  the  mucous  membrane  of  the  pig  or 
rabbit  from  the  deeper  layers  of  the  stomach,  cut 
the  mucous  membrane  into  the  smallest  pieces, 
wash  slightly  with  water,  pour  off  the  water  with 
all  possible  care,  and  cover  the  slightly  moist 
residue  with  glycerine.4  Before  using,  add  dilute 
hvdrochloric  acid. 


1  Eberle  :  Physiologie  der  Verdauung,  1834,  p.  100. 

2  Eberle  :  loc.  cit.,  p.  79. 

3  Dilute  hydrochloric  acid.  —  Add  to  10  c.c.  officinal  HC1, 
sp.  gr.  1.124  (about  25  per  cent  HC1),  enough  water  to  make 
1000  c.c.  This  solution  will  contain  about  0.281  per  cent  HC1. 
(Salkowski's  Practicum,  1893,  p.  130.) 

4  Von  Wittich :  Arcbiv  fur. die  gesammte  Physiologie,  1869, 
ii,  p.  194. 


PROTEID   DIGESTION    BY   PEPSIN  43 

Digestion  -with  Artificial  Gastric  Juice.  —  Pre- 
pare three  flasks,  A,  B,  and  C.  In  A  place  100  c.c. 
artificial  gastric  juice;  in  B,  100  c.c.  0.2  per  cent 
HC1;  and  in  C,  a  piece  of  dried  gastric  membrane 
and  100  c.c.  distilled  water.  In  each  of  the  three 
flasks  place  a  small  piece  of  cooked  meat,  and  keep 
the  flasks  about  five  hours  at  35-40°  C.1  Com- 
pare the  result  with  that  observed  in  natural 
digestion. 

The  artificial  gastric  juice  will  digest  the  meat 
as  did  the  natural  juice  in  the  stomach,  but  neither 
the  acid  alone,  nor  the  mucous  membrane  free 
from  acid,  will  digest.  There  is  a  ferment  in  the 
mucous  membrane,  but  it  will  not  act  except  in 
an  acid  medium.  *' 

Extraction  of  Pepsin.  —  Pepsin  more  or  less  con- 
taminated with  proteid  (pepsin  may  itself  be  a  proteid) 
may  be  precipitated  from  a  glycerine  extract  by  alco- 
hol.2 The  pepsin  may  also  be  carried  down  mechani- 
cally by  an  indifferent  precipitate  as  in  Brticke's 
method,3  in  which  the  mucous  membrane,  acidulated 
with  phosphoric  acid,  is  allowed  to  digest  until  the 
proteids  are  mostly  converted  into  soluble  peptone. 
The  mixture  is  then  neutralized  with  lime  water. 
The  insoluble  calcium  phosphate  thus  formed  falls  as 

1  Eherle  :  loc.  cit. 

2  Von  Wittich:  loc.  cit.,  p.  195. 

3  Briicke  :  Sitzungsberichte  der  konigliche  Akademie  der 
Wissenschaften  zu  Wien,  1862,  xliii,  p.  601. 


44  FERMENTATION 

a  fine  powder  carrying  the  pepsin  with  it.  The  precip- 
itate is  dissolved  in  very  dilute  hydrochloric  acid,  and 
to  this  solution  is  added  a  solution  of  cholesterin  in 
alcohol  and  ether.  When  the  two  solutions  are  mixed, 
the  choiesterki  separates  as  an  abundant,  fine  powder 
bearing  the  pepsin  with  it.  The  cholesterin  is  removed 
with  ether,  leaving  the  pepsin. 

Ammonium  sulphate  may  also  be  used  as  the  me- 
chanical precipitant.1 

Change  of  Proteid  to  Peptone    by  Pepsin.  —  1. 

Place  in  a  test-tube  five  drops  of  the  glycerine 
extract  of  pepsin  with  5  c.c.  0.2  per  cent  hydro- 
chloric acid  and  a  small  piece  of  fibrin.2  Keep 
the  mixture  at  35-40°  C. 

In  a  short  time  the  fibrin  will  be  dissolved. 
Appropriate  tests  will  show  that  it  has  been  con- 
verted to  peptone.  2.  Repeat  the  preceding  ex- 
periment, using  commercial  pepsin  (never  very 
free  from  proteid). 

Splitting  of  Casein  by  Rennin. 

Rennin  Extract.  —  Allow  the  mucous  membrane 
of  the  stomach  (preferably  the  fourth  stomach  of 

1  Kiihne  and  Chittenden:  Zeitschrift  fiir  Biologie,  1886, 
xxii,  p.  428. 

2  Preparation  of  fibrin.  —  With  a  bundle  of  smooth  rods 
whip  blood  as  it  flows  from  an  artery  until  the  fibrin  gathers  on 
the  rods.  Wash  the  fibrin  in  running  water  until  the  red  cor- 
puscles are  removed  and  the  fibrin  shows  its  natural  color. 
Preserve  the  fibrin  in  glycerine. 


SPLITTING   OF   CASEIN    BY   RENNIN  45 

the  suckling  calf)  to  stand"  twenty-four  hours  in 
150-200  c.c.  0.1-0.2  per  cent  solution  of  hydrochlo- 
ric acid.    Then  neutralize  the  acid  with  great  care.1 

Separation  of  Reunin.  —  The  extract  just  prepared 
contains  pepsin  as  well  as  rennin.  The  rennin  may 
be  separated  as  follows.  The  neutralized  extract  is 
repeatedly  shaken  with  fresh  amounts  of  magnesium 
carbonate.  The  resulting  precipitates  carry  down 
almost  all  the  pepsin  and  very  little  rennin.  The 
filtrate  still  rapidly  coagulates  milk,  but  contains  only 
traces  of  pepsin.  This  filtrate  is  now  precipitated 
with  lead  acetate,  the  precipitate  is  decomposed  with 
very  dilute  sulphuric  acid,  and  the  mixture  filtered. 
To  the  filtrate,  which  contains  the  rennin,  is  added  a 
solution  of  stearin  soap  in*  water.  Thereupon  the 
soap  is  thrown  out  of  solution  and  falls,  carrying  the 
rennin  with  it.  The  soap  is  then  removed  by  shaking 
with  ether,  and  the  rennin  remains.2 

Precipitation  of  Casein.  —  Add  1  C  C.  of  the 
neutral  extract  to  25  c.c.  fresh  milk  at  36-38°  C. 
(Xormal  milk  is  amphoteric.  If  the  reaction 
be  acid,  the  acid  should  be  very  carefully 
neutralized.) 

In  a  few  minutes  the  milk  will  separate  into 

1  Hammarsten  :  tfpsala  Lakateforenings  Forhandlingar,  1872, 
viii,  pp.  63-86.  Abstract  by  author  in  Maly's  Jahresberieht 
iiber  die  Fortschritte  der -Thierchemie,  1872,  ii,  pp.  118-125. 

2  Hammarsten  :  Lehrbuch  der  physiologischen  Cnemie,  1S95, 
p.  241. 


46  FERMENTATION 

curd  and  whey.  The  curd  is  casein  together 
with  the  fat  globules  carried  down  as  it  precipi- 
tates. The  whey  is  a  dilute  saline  solution  of 
milk-albumin,  milk  sugar,  etc. 

Test  the  chemical  reaction.  The  mixture  is 
still  neutral.  Milk  may  also  be  curdled  by  acid, 
either  added  artificially  or  produced  in  the  milk 
itself  by  lactic  acid  fermentation  of  milk  sugar. 
The  absence  of  an  acid  reaction  in  the  above  exper- 
iment excludes  precipitation  through  acid  fermen- 
tation of  milk  sugar.  Casein  prepared  free  from 
milk  sugar  is  also  precipitated  by  rennin.  Finally, 
rennin,  extracted  by  the  method  given  above,  does 
not  act  upon  milk  sugar,  but  rapidly  precipitates 
casein. 

Analogy  suggests  that  the  specific  action  of 
the  rennin  may  be  the  splitting  of  casein  and  that 
the  precipitation  may  be  a  secondary  process. 
The  following  experiments  determine  this  matter. 

Experiments  of  Arthus  and  Pages.1  —  Prepare 
two  solutions,  A  and  B. 


A. 
Milk  100  c.c. 

Neutral  oxalate  of 

potassium  1%  5  c.c. 

Rennin  1  to  250        4  c.c. 


B. 

Milk  100  c.c. 

Neutral  oxalate  of 

potassium  1  %         5  c.c. 
Water  4  c.c. 


1  Arthus  and  Pages :  Archives  de  physiologie,  1890,  p.  334. 


SPLITTING   OF   CASEIN   BY   EENNIN  47 

(Rermin,  1  to  250,  is  a  pastille  of  Hansen  dis- 
solved in  250  c.c.  H20.) 

Keep  both  mixtures  at  38°  C.  during  forty 
minutes.  1.  Boil  25  c.c.  from  each  solution. 
Solution  A  coagulates,  while  solution  B  shows 
no  trace  of  coagulation.  Hence  the  action  of 
rennin  has  rendered  the  casein  in  A  coagulable 
on  boiling. 

2.  To  25  c.c.  from  each  solution  add  8  c.c.  of 
a  solution  of  calcium  chloride  capable  of  pre- 
cipitating exactly,  in  equal  volumes,  the  solution 
of  potassium  oxalate.  By  this  addition  the  cal- 
cium oxalate  is  removed  and  the  calcium  chloride 
remains  in  slight  excess. 

A  will  coagulate  ;  B  will  not.  Hence  the  casein 
in  solution  A  has  been  so  changed  by  rennin  that 
it  is  precipitated  on  the  addition  of  a  small  quan- 
tity of  calcium  chloride.  Solution  A  may  also 
be  precipitated  by  restoring  its  original  content 
of  calcium  chloride,  i.e.  by  adding  5  c.c.  of  the 
above  calcium  chloride  solution,  which  will  exactly 
combine  with  the  5. c.c.  of  potassium  oxalate. 

If  small  quantities  of  rennin  be  added  to 
natural  milk  and  equal  portions  of  the  milk  be 
tested  from  time  to  time  by  boiling,  the  amount 
coagulated  will  be  greater  the  longer  the  rennin 
acts.  An  amount  of  calcium  chloride  too  small 
to   produce    coagulation   in   the   early  stages  of 


48  FERMENTATION 

leu niii  action  is  sufficient  to  produce  coagulation 
when  added  in  the  later  stages. 

Evidently,  in  the  clotting  of  milk  by  rennin 
two  separate  phenomena  must  be  distinguished : 
(1)  the  chemical  transformation  of  casein  by 
rennin,  (2)  the  precipitation  of  the  transformed 
casein  by  the  calcium  chloride.  (This  salt  favors 
also  the  splitting  of  the  casein.)  Rennin  may 
therefore  be  classed  with  pepsin  and  trypsin. 

According  to  Hammarsten  the  casein  is  split 
into  phosphorus-free  albumose  and  phosphorus- 
holding  paracasein.  Heat  is  set  free.  It  is  the 
paracasein  which  precipitates.  It  is  less  soluble 
than  casein. 

Precipitation  of  Fibrin  by  Fibrin  Ferment 

Buchanan's  Experiment.  —  Press  blood  clot 
through  a  linen  cloth.  Add  the  liquid  thus  ob- 
tained to  a  serous  fluid,  wfllch  does  not  clot  spon- 
taneously, such  as  ascitic  fluid,  pleural  effusion, 
hydrocele  fluid. 

After  some  hours  a  firm,  translucent  clot  will 
form.1 

Extraction  of  Fibrin  Ferment.  Schmidt's  Method. 
—  Coagulate  one  part  of  serum  from   the  blood 

1  Buchanan  :  London  Medical  Gazette,  1835,  xviii,  p.  51; 
idem,  1845,  xxxvi,  p.  617.  This  discovery  was  first  announced 
in  1831. 


PRECIPITATION    OF   FIBRIN  49 

of  ox,  dog,  or  horse,  by  adding  15-20  parts  strong 
alcohol.  After  at  least  fourteen  days,  filter,  dry 
the  moist  residue  over  sulphuric  acid,  pulverize 
the  dried  substance,  stir  it  with  water  (twice  the 
volume  of  the  serum  originally  taken)  and  after 
allowing  sufficient  time  for  solution,  filter.  The 
filtrate  contains  the  fibrin  ferment.1 

Gamgee's  Method.  —  Allow  freshly  prepared  fi- 
brin (obtained  by  washing  a  blood  clot  free  from 
corpuscles)  to  stand  three  days  in  8.0  per  cent 
solution  of  sodium  chloride.     Filter.2 

The  filtrate  is  rich  in  fibrin  ferment. 

Extraction  of  Fibrinogen.  —  Eeceive  three 
volumes  of  blood  directly  from  an  artery  into 
one  volume  of  saturated  solution  of  magnesium 
sulphate,  which  will  prevent  the  blood  from 
clotting.  Separate  the  corpuscles  from  the  liquid 
plasma  by  the  centrifugal  machine.  Add  to  the 
plasma  an  equal  volume  of  saturated  solution  of 
sodium  chloride.  Flakes  of  fibrinogen  will  be 
precipitated.  Filter  as  quickly  as  possible,  for 
that  purpose  dividing  the  liquid  among  several 
funnels  each  with  a  folded  filter  paper.  Press 
the  filter  papers  containing  the  residue  between 
fresh  filter  paper,  in  order  to  remove  the  adherent 

1  Schmidt :  Archiv  fur  die  gesammte  Physiologie,  1872,  vi, 
p.  457. 

2  Gamgee  :  Journal  of  physiology,  1S79,  ii,  p.  151. 

4 


50  FERMENTATION 

liquid.  Tear  the  filter  containing  the  fibrinogen 
into  small  pieces.  Dissolve  the  fibrinogen  which 
sticks  to  the  filter  as  a  tough,  elastic  mass,  in  a 
quantity  of  8  per  cent  sodium  chloride  solu- 
tion equal  to  about  one-third  the  quantity  of  the 
magnesium  sulphate  solution  originally  taken. 
Filter  off  the  fragments  of  paper.  Purify  by 
reprecipitation  with  an  equal  volume  of  saturated 
solution  of  sodium  chloride.  Filter.  Dry  as 
before,  and  add  a  small  quantity  of  water  to  the 
finely  divided  filter  to  which  the  precipitate 
clings.  This  water  will  take  a  small  quantity  of 
salt  from  the  precipitate,  and  in  this  dilute  saline 
solution  the  fibrinogen  will  dissolve.1 

Precipitation  of  Fibrinogen  by  Fibrin  Ferment. — 
Add  to  the  dilute  saline  solution  of  fibrinogen  a 
solution  containing  fibrin  ferment. 

Fibrin  will  form. 

Ammoniacal  Fermentation  of  Urea  by 
Urease 

1.  Place  100  c.c.  fresh  human  urine  in  each 
of  three  clean  flasks  marked  A,  By  C.  To  B  and 
C  add  1  c.c.  of  urine  that  has  become  ammoniacal 
upon  standing  in  the  atmospheric  air.     Add  also 

1  Hammarsten  :  Arehiv  fur  die  gesaramte  Physiologie,  1879, 
xix,  p.  563.  Also  idem,  1880,  xxii,  p.  431.  Hammarsten's  first 
publication  was  in  Nova  acta  regia  societas  scientiarum  Upsali- 
eusis,  1873,  (3),  ix. 


FERMENTATION  OF  UEEA  BY  UREASE     51 

to  C  2  per  cent  of  a  saturated  solution  of  carbolic 
acid  in  water.  Let  B  and  C  stand  in  a  warm 
place  sixteen  days. 

2.  Withdraw  5  c.c.  from  flask  A.  Note 
whether  the  urine  is  clear  or  turbid,  and 
whether  it  effervesces  on  the  addition  of  a 
dilute  acid.  Withdraw  2  c.c.  from  flask  A  and 
determine  its  percentage  of  urea  by  the  hypo- 
bromite  method. 

Centrifugalize  a  portion  of  the  remaining  con- 
tents of  flask  A.  With  a  microscope  examine 
the  sediment  for  crystals  of  ammonio-magnesium 
phosphate  and  for  micro-organisms,  especially  the 
micrococcus  urea?,  which  Occurs  in  long  curved 
chains  of  round  cells  about  1.5  /jl  in  diameter. 

3.  After  sixteen  days  repeat  these  observa- 
tions on  the  urine  in  flasks  B  and  C.  Record 
the  results  obtained  from  all  three  flasks  in  the 
table  on  page  52. 

The  table  shows  that  the  hydrolysis  of  urea 
into  ammonium  carbonate  still  takes  place  in 
urine  containing  enough  carbolic  acid  to  destroy 
the  micro-organisms  long  known  to  be  the  cause 
of  the  ammoniacal  fermentation.1  It  is  therefore 
probably  due  to  a  ferment,  which  escapes  from 
the  cells  after  their  death. 

1  Hoppe-Seyler  :  Medicinisch-chemische  Untersuchungen, 
Berlin,  1866,  p.  570. 


52 


FERMENTATION 


v3 

£.! 

ej    - 

S  it 
O 

06  |  « 

Per  cent 

of 

Urea. 

Reaction 

to 
Acids. 

Clear 

or 

Turbid. 

Content 

of 
Carbolic 

Aeid. 

go 
at) 

A 

Normal 

B 

Septic 

C 

Aseptic 

FERMENTATION  OF  UREA  BY  UREASE     53 

Prior  to  1860  ammoniacal  decomposition  of 
urine  was  vaguely  classed  as  a  fermentation.  In 
that  year  Miiller1  suggested  that  it  might  be  due 
to  a  body  like  beer-yeast.  In  1862  Pasteur2 
discovered  such  a  yeast,  which  he  called  Torula 
urece.  Colin  first  classed  it  with  the  micrococci. 
It  is  aerobic.  Miguel  finds  seven  species  of 
bacilli,  nine  micrococci,  and  one  sarcina,  that 
decompose  urea.  These  obtain  their  nitrogen 
ordinarily  from  proteids,  but  in  the  absence  of 
proteids  may  utilize  urea. 

Extraction  of  Urease.  —  To  10  C.C.  of  urine 
undergoing  an  active  ammoniacal  fermentation, 
add  50  c.c.  of  strong  alcohol,  and  allow  the 
mixture  to  stand  in  a  well-corked  flask.  After 
five  days  place  the  precipitate  upon  a  very  small 
filter  and  wash  it  with  50  c.c.  of  fresh  alcohol. 
(Preserve  both  filtrates  for  recovery  of  the  alcohol 
by  redistillation.) 

1.  Add  a  very  small  quantity  of  this  precip- 
itate to  a  neutral  2  per  cent  solution  of  urea. 
Test  the  reaction.  Place  the  mixture  in  a  water 
bath  at  38°  C. 

After  a  few  minutes  again  test  the  reaction. 

It  will  be  strongly  alkaline. 

1  Miiller:  Journal  fur  praktische  Chemie,  1860,  Ixxxi,  p.  467. 

2  Pasteur  :  Comptes  vendus  de  l'academie  des  sciences,  Paris, 
1860,  1,  p.  869.     See  also  Van  Tieghern,  idem,  1864,  p.  210. 


54  FERMENTATION 

After  a  short  time  the  odor  of  ammonia  will  be 
perceptible.  The  alcoholic  precipitate  contains  a 
ferment  capable  of  quickly  hydrating  urea. 

"The  alcoholic  precipitate  from  the  unfiltered 
urine  consists  chiefly  of  various  salts  together 
with  the  cells  of  the  Torula,  hence  when  treated 
with  water  some  of  the  salts  are  dissolved  and 
pass  with  the  ferment  through  the  filter.  If  this 
first  aqueous  extract  be  again  precipitated  with 
alcohol,  a  portion  of  the  salts  will  be 'again 
removed,  and  if  this  second  precipitate  be  several 
times  redissolved  in  water  and  reprecipitated 
with  alcohol,  the  body  with  the  ferment  proper- 
ties may  be  ultimately  separated  —  as  an  amor- 
phous white  powder  soluble  to  a  clear  solution 
in  distilled  water  and  not  characterized  by  any 
special  chemical  reactions." 

The  ferment  is  not  secreted  by  the  cells  into 
the  surrounding  liquid,  but  is  retained  within  the 
cell  bodies,  for  the  living  cells  may  be  filtered  off, 
and  the  filtrate  will  not  hydrate  the  urea.1 

Splitting  and  Synthesis  of  Fats 

Chemistry  of  Fats  and  Soaps.  —  When  olive  oil 
is  saponified,  glycerine   appears  (Scheele,  1779). 

1  Lea  :  Journal  of  Pl^siology,  1885,  vi,  p.  138.  See  also 
Musculus :  Comptes  rendus  de  l'academie  des  sciences,  Paris, 
187-1,  lxxviii,  p.  132  ;  idem,  1876,  lxxxii,  p.  333  ;  Archiv  fiir 
die  gesammte  Physiologie,  1876,  xii,  p.  214. 


SPLITTING   AND    SYNTHESIS    OF   FATS  00 

It  is  related  to  the  alcohols  (Chevreul,  1813), 
being  a  compound  ether  or  ester,  a  combination 
of  an  alcohol  with  an  acid.  Commercially  gly- 
cerin 3  is  prepared  by  exposing  neutral  fats,  such 
as  stearin,  to  superheated  steam,  whereby  the 
neutral  fat  is  split  into  glycerine  and  fatty  acid. 


CHoO 

1 

•CO(CH2)16 

•CH3 

H 

•OH 

CHO 

•CO(CH2)16 

•CH3 

+ 

H 

•OH 

= 

CH20 

•CO(CH2)16 

■CH3 

H 

•OH 

STEARIN 

WATER 

CH2 

i 

•OH 

CO 
1 

•OH(CH2)16-CH 

1 
CH 

i 

•OH 

+ 

1 

CO 
1 

•OH(CH,)16'CH 

CH2 

•OH 

1 
CO 

•OH(CH2)16-CH 

GLYCERINE 

STEARIC    ACID 

If  an  alkali  be  present,  it  will  combine  with 
the  fatty  acid  to  form  a  soap. 


CO 

•OH(CH.2)16-CH3 

Xa  •  OH 

CO 

■OH(CH2)16-CH3 

+         Xa  •  OH         = 

CO 

•OH(CH2)16-CH3 

STEARIC    ACID 

Xa  •  OH 

SODIUM 
HYDROXIDE 

CO-OXa(CH2)16-CH3 

H-OH 

CO-OXa(CH2)16-CH3 

+ 

HOH 

CO-OXa(CH2)16.CH3 

H-OH 

SODIUM    STEAEATE 

WATER 

Splitting  of  Fats  by  the  Pancreatic  Juice.      Ber- 
nard's Experiment  —  Place   2  c.c.  neutral  olive 


56  FERMENTATION 

oil  in  a  test-tube  and  add  a  small  quantity  of 
pancreatic  juice  (or  a  piece  of  fresh  pancreas  or 
extract  of  pancreas).  Test  the  reaction  of  the 
mixture.  It  is  alkaline.  Note  that  a  white, 
creamy  liquid  forms  almost  immediately.  This 
"  emulsion  "  is  composed  of  a  multitude  of  small 
fat  globules. 

Test  the  reaction  again.  It  gradually  becomes 
acid. 

It  is  evident  that  under  the  influence  of  the 
pancreatic  juice  the  fatty  matter  is  not  simply 
finely  divided  and  emulsified,  but  that  it  has  also 
been  modified  chemically.1 

In  order  to  study  the  splitting  of  neutral  fats 
by  lipase,  a  ferment  found  in  the  pancreatic  juice, 
it  is  necessary  (1)  to  prepare  a  perfectly  neutral 
fat,  and  (2)  to  recognize  the  fatty  acid  as  soon  as 
it  is  set  free. 

Preparation  of  Neutral  Fat.  —  Shake  commer- 
cial olive  oil  (which  always  contains  fatty  acid) 
for  two  hours  at  95°  C.  in  a  separating  funnel 
with  a  saturated  solution  of  barium  hydroxide. 
Allow  the  mixture  to  stand  until  the  oil  sepa- 
rates from  the  hydroxide.  Eemove  the  hydrox- 
ide.    Filter  the  oil. 

The    Emulsion    Test   for   Fatty   Acid.      BriicJie's 

1  Bernard  :  Comptes  rendus  de  l'academie  des  sciences,  Paris, 
1849,  xxviii,  p.  250. 


SPLITTING    AND    SYNTHESIS    OF    FATS  57 

Experiment.  —  1.  Shake  1  c.c.  neutral  olive  oil 
in  a  test-tube  with  5  c.c.  0.25  per  cent  sodium 
carbonate  solution. 

The  oil  will  be  broken  up  into  large  globules 
which  will  speedily  reunite,  leaving  the  liquid 
clear. 

2.  Shake  1  c.c.  rancid  olive'  oil  (containing 
about  5.5  per  cent  fatty  acid)  with  5  c.c.  0.25  per 
cent  sodium  carbonate  solution. 

The  mixture  becomes  instantly  milky.  The 
oil  is  divided  into  globules  of  microscopic  size. 
The  emulsion  is  permanent. 

3.  Shake  1  c.c.  neutral  olive  oil  with  5  c.c.  water. 
The  water  and  oil  will  not  mix. 

4.  Shake  1  c.c,  neutral  oil  with  water  con- 
taining  soap. 

The  oil  will  be  emulsified.  It  is  probable 
therefore  that  soap  contributes  to  the  emulsion, 
perhaps  by  coating  the  fine  particles  of  oil  with  a 
membrane  that  prevents  their  reunion.1 

Gad's  Experiment.  —  1.  Fill  a  watch  glass 
about  5  cm.  in  diameter  with  0.25  per  cent  solu- 
tion of  sodium  carbonate.  With  a  glass  rod 
carefully  place  a  large  drop  of  rancid  olive  oil 
(containing  5.5  per  cent  fatty  acid)  upon  the 
surface  of  the  soda  solution. 

1  Briioke  :  Sitzungsbeiichte  der  kaiserlichen  Akademie  der 
Wissenschaften  zu  Wien,  1870,  lxi,  pp.  613-61-4. 


58  FERMENTATION 

The  drop  will  come  to  rest,  and  for  a  moment 
both  the  drop  and  the  surrounding  liquid  remain 
clear.  Very  soon,  however,  the  oil  is  covered 
with  a  white  layer,  and  through  the  soda  solu- 
tion spreads  a  white  cloud  which  becomes  denser 
and  denser  until  the  oil  drop,  steadily  diminish- 
ing in  size,  floats  in  a  milky  white  liquid. 

2.  Eepeat  the  experiment,  observing  the  oil 
drop  under  a  low  power  of  the  microscope. 

Note  the  extraordinary  motion  in  the  neigh- 
borhood of  the  oil  drop,  and  how  the  particles  of 
oil  are  thrown  out  in  strong  eddies. 

3.  Examine  the  completed  emulsion  under  a 
higher  power  of  the  microscope. 

There  appear  exceedingly  small  fat  drops  of 
very  uniform  size.  The  milky  fluid  is  the  finest 
and  most  uniform  emulsion.1 

BachforcFs  Experiment.  —  "  Arrange  a  series  of 
watch  glasses  containing  0.25  per  cent  solution 
sodium  carbonate.  Place  in  a  test-tube  2  c.c. 
neutral  olive  oil  and  1  c.c.  pancreatic  juice  (or 
extract).  Shake  the  tube  and  allow  the  juice 
and  oil  to  separate,  then  pipette  a  drop  of  oil 
from  the  surface  and  place  it  on  the  soda  solu- 
tion in  watch  glass  1.  Again  shake  the  tube 
and  allow  the  oil  and  juice  to  separate,  then 
pipette  as  before,  placing  a  drop  of  oil  in  watch 

1  Gad:  Archiv  fur  Physiologie,  1878,  p.  183. 


SPLITTING   AND    SYNTHESIS    OF   FATS  59 

glass  2.  Again  shake  and  pipette  as  before,  and 
repeat  this  process  every  three  or  four  minutes 
until  the  experiment  is  completed.  The  begin- 
ning of  the  experiment  and  the  time  of  each 
pipetting  must  be  carefully  noted.  If  the  pipet- 
tings  are  three  minutes  apart,  then  the  first  drop 
of  oil  will  have  been  exposed  three  minutes  to 
the  action  '  of  the  pancreatic  juice,  the  second 
drop  six  minutes,  the  third  nine  minutes,  and 
so  on.1 

The  gradual  increase  in  fatty  acid  will  be 
shown  by  the  gradual  increase  in  the  amount 
of  the  spontaneous  emulsion.2 

It  has  just  been  shown  that  lipase  will  hydro- 
lyze  neutral  fats  into  fat^y  acid  and  glycerine. 
We  must  now  enquire  whether  this  ferment 
ean  effect  the  synthesis  of  fats,  in  other  words 
whether  its  action  is  reversible.     For  this  pur- 

1  Rachford:  Journal  of  physiology,  1891,  xii,  p.  SI.  Rach- 
ford  used  J  c.c.  fresh  pancreatic  juice  obtained  by  placing  a 
glass  tube  in  the  pancreatic  duct  of  the  rabbit  (see  page  80). 

2  "  There  is  a  possible  error  in  this  method  which  had  better 
be  spoken  of  here.  It  would  seem  that  the  alkali  of  the  pan- 
creatic juice  would  combine  with  the  fatty  acids  forming  soap, 
and  in  this  way  the  oil  would  soon  be  emulsified  in  the  juice 
itself  and  not  separate  after  shaking.  This  would  indeed  be  a 
serious  drawback  if  it  actually  occurred,  but  in  truth  it  does  not 
occur  until  late  in  the  experiment  after  we  have  obtained  the 
information  we  have  sought  by  the  spontaneous  emulsion 
method."     (Rachford,  loc.  cit.,  p.  82). 


60  FERMENTATION 

pose  an  extract  of  lipase  may  be  used,  first,  to 
split  a  neutral  fat  (or  glycerol  ester)  into  its  con- 
stituent fatty  acid  and  alcohol  (glycerine  is  a 
trihydric  alcohol),  and  second,  to  form  a  neutral 
fat  from  fatty  acid  and  alcohol. 

Extraction  of  Lipase.  From  Pancreas.  —  Re- 
move the  pancreas  of  the  pig  within  thirty  min- 
utes after  the  death  of  the  animal.  Dissect  off 
as  much  of  the  fat  as  possible.  Reduce  the  pan- 
creas to  a  fine  pulp  in  a  mortar  with  coarse  well- 
washed  white  sand.  Extract  the  lipase  with  a 
little  water  or  glycerine. 

From  Liver.  —  Remove  the  liver  of  the  pig 
within  thirty  minutes  of  the  death  of  the  animal. 
Reduce  50  gms.  to  a  fine  pulp  in  a  mortar  with 
about  200  c.c.  water.  Filter.  Dilute  the  watery 
extract  to  500  c.c. 

Hydrolysis  of  Ethyl  Butyrate  by  Lipase.  — 
Place  in  each  of  two  test-tubes,  A  and  B,  4  c.c. 
water,  0.1  c.c.  toluene,1  and  0.26  c.c.  ethyl  buty- 
rate.2 Cork  the  tubes  tightly.  Place  them  in  the 
water  bath  for  five  minutes,  to  bring  them  to 
the  temperature  of  the  bath,  40°  C.     Add  1  c.c.  of 

1  Toluene  is  an  antiseptic,  which  prevents  the  splitting  of 
the  neutral  fat  by  bacteria. 

2  Ethyl  butyrate  hydrolyzes  more  rapidly  than  butter  fat. 
It  has  the  further  advantage  that  the  amount  split  by  the 
temperatures  employed  during  the  time  of  the  experiment  is  too 
small  to  be  measurable. 


SPLITTING    AND    SYNTHESIS    OF   FATS  61 

the  aqueous  extract  of  lipase  to  each.  Boil  tube 
B.  Place  both  tubes  at  40°  C.  for  fifteen  min- 
utes. Eemove  them  from  the  bath  and  plunge 
them  into  ice-water  (to  check  further  ferment 
action).  Titrate  with  ^  KOH,  using  neutral  lit- 
mus as  the  indicator.1     The  initial  acidity  of  the 

1  A  normal. solution  contains  in  each  litre  one  equivalent 
weight  of  the  active  substance,  i.e.  that  mass  of  the  active  sub. 
stance  which  is  equivalent  to  the  atomic  weight  of  a  univalent 
element  in  the  reaction  for  which  the  normal  solution  is  to  be 
employed.  Equal  volumes  of  different  normal  solutions  are 
equivalent  to  each  other.  Thus,  1  c.c.  normal  alkali  solution 
requires  for  neutralization  exactly  1  c.c.  normal  acid,  no  matter 
what  acid  is  employed  to  make  the  normal  solution. 

Preparation  of  Normal  Potassium  Solution. — The  content  of 
KOH  in  1  litre  is  56.16  grams.  Dissolve  60  gms.  purest  com- 
mercial KOH  (which  always  contains  considerable  water)  in  a 
graduated  cylinder  in  about  950  c.c.  water.  Determine  the 
true  content  of  KOH  by  titration  with  a  normal  oxalic  acid 
solution  (prepared  by  dissolving  its  equivalent  weight  63  gms. 
in  1  litre  water)  as  follows.  Thoroughly  stir  the  potassium  hy- 
droxide solution,  fill  a  burette  with  a  portion  of  the  well-mixed 
solution.  Place  10  c.c.  normal  oxalic  acid  solution  in  a  beaker 
and  add  a  few  drops  of  solution  of  rosolic  acid  as  indicator. 
Add  the  alkali  from  the  burette  cautiously  until  the  end  point 
of  the  reaction  is  reached,  i.  e.  until  the  indicator  gives  a  red 
color  which  does  not  quickly  disappear.  As  10  c.c.  of  acid  solu- 
tion should  exactly  neutralize  10  c.c.  of  alkali  solution,  pro- 
vided both  were  normal,  it  follows  that  the  quantity  of  KOH 
solution  necessary  to  neutralize  is  to  10  c.c.  as  the  total  quantity 
of  the  original  KOH  solution  is  to  x.  x  will  be  the  number  of 
cubic  centimetres  to  which  the  KOH  solution  must  be  diluted 
in  order  to  make  it  normal.  A  portion  of  the  normal  solution 
should  then  be  diluted    1:20,    and   preserved    in  an  air-tight 


62  FEKMENTATION 

enzyme  solution,  usually  0.1  to  0.2  c.c.  ^o  KOH, 
should  be  deducted  from  the  cubic  centimetres 
KOH  required  to  neutralize  the  fatty  acid 
formed.1 

Fatty  acid  will  appear  in  tube  A,  but  not  in 
tube  B,  in  which  the  enzyme  was  destroyed  by 
boiling. 

Synthesis  of  Neutral  Fat  by  Lipase.  —  1.  Place 
5  c.c.  T^7  butyric  acid,  2  c.c.  13  per  cent  alcohol, 
1  c.c.  diluted  glycerine  extract  of  pig's  pancreas 
(or  aqueous  extract  of  liver)  in  each  of  two  test- 
tubes,  A  and  B.  Boil  the  contents  of  test-tube 
B.  Seal  both  tubes.  Keep  them  thirty-six 
hours  at  48.5°  C. 

On  opening  the  tubes,  A  will  give  a  distinct 
odor  of  ethyl  butyrate ;  none  will  be  found  in  B, 
in  which  the  ferment  was  destroyed  by  boiling.2 

2.  Place  5  gms.  glycerine,  2  gms.  isobutyric 
acid,  125  gms.  water,  1  c.c.  neutralized  blood  serum 
(or  aqueous  extract  of  pig's  liver)  in  each  of  two 


flask.     (Compare   Miiller   and  Kiliani:    Kurzes   Lehrbuch   der 
analytischen  Cheinie,  1900,  p.  31  and  p.  83). 

At  30°  (summer  temperature)  0.26  c.c.  ethyl  butyrate  weighs 
0.2300  gram.  This  quantity,  if  completely  hydrolyzed,  would 
require  39.7  c.c.  ^  KOH. 

1  Kastle  and  Loevenhart :  American  chemical  journal,  1900, 
xxiv,  pp.  491-525.  Also  Loevenhart :  American  journal  of 
physiology,  1902,  vi,  pp.  331-350. 

2  Kastle  and  Loevenhart :  loc.  cit.,  p.  518. 


SPLITTING   AND    SYNTHESIS    OF   FATS  63 

test-tubes,  A  and  B.  Boil  the  contents  of  tube 
B.  Place  both  at  37°  C.  At  intervals  of  half 
an  hour  titrate  a  portion  from  each  tube  with 
2*0  KOH  solution.  The  acidity  will  diminish  in 
both,  but  much  more  rapidly  in  the  tube  contain- 
ing the  active  ferment. 

'  The  acidity  is  diminished  by  the  combination 
of  the  fatty  acid  with  the  glycerine  to  form  a 
neutral  fat.1 

Fats  are  hydrolyzed  to  some  extent  in  the  stomach,2 
but  stomach  lipase  is  active  only  in  neutral  solutions. 
It  is  inhibited  or  destroyed  by  0.3  per  cent  hydro- 
chloric acid.  Other  ethereal  salts  besides  the  fats  are 
hydrolyzed  in  the  intestine,  e.  g.  salol.3 

The  rate  of  change  by  lipase  increases  with  the 
amount  of  the  enzyme  present.4 

Reversible  action  is  seen  in  ferments  other  than 
lipase,  as  in  the  following  experiments. 

Splitting  of  Hippuric  Acid  by  Histozyme. —  A  pig's 
kidney  was  perfused  four  hours  with  one  litre  defibri- 
nated    pig's    blood   to  which  0.8   gram  hippuric  acid 

1  Hanriot :  Coniptes  rendus  de  la  societe  de  biologie,  1901, 
p.  70. 

2  Marcet:  Proceedings  Royal  Society,  London,  1858,  ix, 
p.  306.  Ogata  :  Archiv  fur  Physiologic,  1881,  p.  515.  Cash  : 
Archiv  fiiv  Physiologie,  1880,  p.  323. 

3  Baas:  Zcitschrift  fur  physiologische  Chemie,  1S90,  xiv, 
p.  416. 

4  Kastle  and  Loevenhart :  loc.  cit,  p.  511. 


64  FERMENTATION 

(sodium  salt)  had  been  added.  The  hlood  passed 
through  the  kidney  9-10  times. 

Upon  analysis,  there  appeared  0.037  gram  benzoic 
acid,  produced  from  0.1 276  gram  hippuric  acid. 

Synthesis  of  Hippuric  Acid  by  Histozyme.  —  A  pig's 
kidney  was  perfused  three  hours  with  one  litre  defibri- 
nated  pig's  blood  containing  a  neutral  solution  of  0.5 
gram  benzoic  acid  and  0.6  gram  glycocoll.  The  blood 
passed  ten  times  through  the  kidney. 

Found  :  94  mgm.  hippuric  acid.1 

These  actions  depend  upon  a  ferment,  histozyme, 
extracted  by  Schmiedeberg. 

Some  hypothetical  considerations  will  be  of  value 
here.  Compounds  of  carbon  may  be  divided  into 
those  in  which  the  carbon  atoms  are  arranged  in  an 
open    chain,  for  example  ethane,  C2He 

H    II 

I       I  - 
H— C— C— H 

I       I 
H   H 


and  those  in  which  the  chain  is  closed  to  form  a  "car- 
bon ring,"  for  example,  benzene,  C6H6,  which  consists 
of  six  carbon  atoms,  in  a  closed,  ring-shaped  chain,  the 
"  benzene  nucleus,"  with  a  hydrogen  atom  joined 
to  each  carbon  atom  by  its  fourth  affinity  (Kekule, 
1865). 

1  Schmiedeberg  :  Arehiv  fur  experimentelle  Pathologie  und 
Pharmakologie,  1881,  xiv,  pp.  382-383. 


'O* 


SPLITTING   AND    SYNTHESIS    OF  FATS  65 

H  H 

\  /  \  / 

0  =  C"  C  =  C 

/  \  /  \ 

C  C-  H-C  C-H 

^         //  %         //    ■ 

c  -  c  c  -  c 


BENZENE   NUCLETT 
OR   KING 


H  H 

BENZENE 


The  benzene  ring  is  not  easily  opened,  but  deriva- 
tives of  benzene  inay  be  readily  obtained  by  replacing 
hydrogen  atoms.  Thus,  in  aniline  or  amido-benzene, 
C6H5.NH2,  one  hydrogen  atom  is  replaced  by  amide 
radical ;  in  carbolic  acid,  or  phenol.  C6H5.OH,  by 
hydroxyl ;  in  toluene  or  methyl  benzene,  C6H5.CH3, 
by  the  radical  CH3.  The  usarbon  atom  in  methyl 
benzene  is  not  a  part  of  the  benzene  ring,  but  is 
chained  to  the  side  of  the  ring.  The  hydrogen  atoms 
in  the  side-chain  differ  in  their  affinities  from  those 
attached  to  the  ring ;  the  hydrogen  in  the  ring  may 
be  replaced  by  groups  («.^N02)  which  will  not  readily 
replace  the  hydrogen  of  the  side-chain.  This  is  a 
matter  of  special  interest  in  relation  to  the  specific 
action  of  poisons,  ferments,  etc.  By  substituting 
hydroxyl  for  the  hydrogen  of  the  side-chain,  benzyl 
alcohol,  C6H5.CH2.OH,  is  formed.  By  introducing 
carboxyl,  benzoic  acid,  C6H5.CO.OH,  is  obtained.  It 
has  been  shown  above  that  benzoic  acid  and  glyco- 
coll  are  united  in  the  kidney  to  form  hippuric  acid. 
Glycocoll  is  amido-acetic  acid,  CH2(XH2).CO.OH.     It 


66  FERMENTATION 

unites  with  benzoic  acid  by  replacing  the  hydroxyl  in 

the  side-chain,  thus  forming 

C6H5.CO.NH. 

;ch2 

CO.OH 

HIPPUKIC   ACID 

Cinnamic  acid,  toluene,  and  other  aromatic  substances 
are  similarly  excreted  as  hippuric  acid  when  taken 
internally. 

The  reversible  action  of  the  kidney  ferment  is  im- 
portant in  hastening  the  establishment  of  the  equi- 
librium between  benzoic  acid  and  glycocoll.  If  these 
two  bodies  pass  through  the  kidney,  a  certain  amount 
of  hippuric  acid  is  formed ;  if  hippuric  acid  itself 
passes  through  the  kidney,  a  certain  quantity  is  hy- 
drolyzed. 

Relation  of  Reversible  Action  to  Absorption  of  Fat. — 
"Pancreatic  juice  is  capable  of  hydrolyzing  all  the  fat 
of  a  fatty  meal  in  the  period  of  pancreatic  digestion. 
In  the  living  intestine  the  hydrolysis  should  be  com- 
plete, inasmuch  as  the  removal  of  the  products  of  the 
hydrolysis  by  absorption  prevents  the  establishment 
of  equilibrium.  On  the  other  hand,  the  products  of 
the  hydrolysis  in  their  transition  through  the  epithelial 
cells  come  in  contact  with  a  lipolytic  enzyme,  the  pres- 
ence of  which  in  these  cells  has  been  demonstrated  in 
the  above. 

"The  lipase  now  finds  itself  in  contact  with  only 
fatty  acid  and  glycerine,  and  hence  in  acting  catalyti- 
cally  to  bring  about  the  chemical  equilibrium,  it  effects 


IMMUNITY  67 

the  synthesis  of  a  fat.     This  would  offer  a  satisfactory 

explanation  of  the  presence  of  fat  granules  in  these 
cells.  As  the  fatty  acid  and  glycerine  diffuse  out  of 
the  cells  through  the  basement  membrane,  the  fat 
in  these  cells  would  speedily  disappear  were  it  not  that 
these  substances  were  constantly  being  absorbed  from 
the  lumen  of  the  intestine.  "When  absorption  ceases, 
however,  the  fat  present  is  at  once  hydrolyzed  by  the 
lipase  present.  This  hydrolysis  is  in  all  probability 
complete  for  the  reason  that  the  products  of  the 
hydrolysis,  viz.,  glycerine  and  fatty  acid,  are  being 
constantly  removed  by  diffusion.  According  to  this 
view,  therefore,  no  fat  ever  enters  or  leaves  the  epi- 
thelial cells  as  such,  but  as  fatty  acid  and  glycerine. 

'•'These  two  substances  then  enter  the  central 
lacteal,  where  equilibrium  is  again  established  ami 
there  is  a  large  production  of  fat."1 

Immunity 

Ehrlich's  Ricin  Experiments.2  —  Powder  Albert 
biscuits  weighing  6.75  grams.     Add  to  each  cake 

1  Kastle  and  Loevenhart :  loc  cit.,  p.  522. 

2  Ehrlieh  :  Deutsche  medicinische  "Wochensehrift,  1891, 
xvii,  pp.  976-979. 

Ricin  is  a  toxalbumin  extracted  from  the  seeds  of  the  castor 
oil  plant.  It  is  poisonous  in  the  slightest  traces.  Weight  for 
weight  it  is  a  billion  times  more  poisonous  than  corrosive  sub- 
limate. Intravenous  injection  of*  0.03  milligram  (0.00003  gram) 
per  kilo  of  body  weight  is  fatal.  One  gram  commercial  ricin 
would  kill  one  and  one-half  million  guinea-pigs.  The  effect  is 
about  one  hundred  times  less  when  taken  by  the  mouth,  yet 


68  FERMENTATION 

3.2-3.5  c.c.  of  water  containing  ricin.  The  be- 
ginning content  of  ricin  should  be  0.02  gm.  ricin 
for  each  cake  ;  0.035  gm.  is  fatal  in  the  course 
of  five  or  six  clays.  Mix  the  biscuit  powder  and 
ricin  solution  to  a  stiff  dough,  roll  the  dough  into 
rods,  divide  them  into  equal  lengths,  and  dry 
the  portions  quickly  on  a  wire  sieve.  Determine 
the  effect  on  white  mice  of  successively  increas- 
ing doses,  as  follows : 


)AY 

DOSE 

DAY 

DOSE 

1 

0.002  gm. 

9 

0.02 

2 

.   .   . 

10 

0.03 

3 

0.006 

11 

0.04 

4 

0.008 

12 

0.05 

5 

.   .   . 

13 

0.06 

6 

0.01 

14 

.   .   . 

7 

0.0125 

15 

0.08 

8 

0.015 

16 

0.01 

On  the  17th  day  inject  subcutaneously  a  fresh 
mouse  with  the  fatal  dose  —  1  c.c.  of  a  ^o^o o"o"  so" 
lution  per  20  gm.  of  mouse.     At  the  same  time 

even  thus  0.18  gram  will  kill  a  full-grown  man.  The  cause  of 
death  is  agglutination  of  red  blood  corpuscles,  and  hence 
multiple  thrombosis,  especially  of  the  abdominal  vessels. 
Clinically,  violent  diarrhoea  and  progressive  exhaustion  are  ob- 
served. The  toxicity  is  greatly  dependent  on  species.  Guinea- 
pigs  are  far  more  susceptible  than  white  mice.  With  white 
mice  the  fatal  subcutaneous  injection  is  1  c.c.  of  a  solution  con- 
taining zthjW  ricin  l)er  20  grams  °f  Dody  weight. 


IMMUNITY  69 

inject   the   immunized    mice    with   a    dose    one 
hundred  times  as  great.1 

Observe  the  non-immune  and  the  immune  mice 
for  several  days  and  note  the  results. 

Ehrlieh  continued  the  above  experiment  until  the 
immunized  mouse  received  daily  0.5  gm.  of  the  ricin 
by  the  mouth.  Such  animals  bore  safely  subcutaneous 
injections  of  -1q  and  even  more.  The  immunity  also 
appeared  in  that  solutions  of  0.5-1.0  per  cent  applied 
to  the  eyes  of  non-immune  mice  caused  violent  pano- 
phthalmitis, while  immune  mice  bore  easily  the  appli- 
cation of  10  per  cent  solutions. 

This  absolute  local  immunity  was  fully  established 
when  the  general  immunity  had  attained  only  a 
middle  grade.  Normally  the  subcutaneous  injection 
of  400V00  ricin  solution  causes  severe  local  inflamma- 
tion, but  thoroughly  immunized  animals  bear  TisW* 
Quantitative  experiments  show  that  the  resistance  to 
the  poison  is  not  increased  during  the  first  four  days, 
and  the  increase  is  doubtful  on  the  fifth  day,  but  on 
the  sixth  day  a  relatively  high  (for  example  thirteen- 
fold)  general  immunity  is  suddenly  established.  The 
sudden  fall  toward  normal  temperature  observed  in 
diseases  with  a  "crisis,"  such  as  pneumonia,  may  de- 
pend on  the  "  critical  "  establishment  of  immunity. 

Immunity  is  not  increased  by  continued  administra- 
tion of  the  same  dose,  day  by  day.  An  equilibrium 
.appears  to  be  established. 

1  The  mice  in  these  experiments  must  be  carefully  protected 
against  cold  and  wettincr. 


70  FERMENTATION 

The  immunity  once  established  endures  a  consider- 
able time  ;  six  months  and  possibly  much  longer. 

Ricin  Antitoxine.  —  Defibrinate  the  blood  of  the 
immunized  mice.  Divide  it  into  two  portions. 
1.  To  one  portion  add  ricin  solution  in  such  a 
ratio  that  the  mixture  shall  contain  yomIFO'  *■  e- 
twice  the  fatal  amount. 

Inject  a  fresh  mouse  subcutaneously  with  1  c.c. 
of  this  mixture  per  20  grams  of  weight. 

The  poison  will  be  borne.  It  has  been  neu- 
tralized by  the  serum  of  the  immune  animal. 
This  result  accords  with  the  discovery  of  Behring 
and  Kitasato  that  immunity  in  diphtheria  and 
tetanus  depends  on  the  power  of  the  serum  to 
neutralize  the  poison. 

2.  Divide  the  second  portion  of  the  antitoxine 
blood  among  six  small  test-tubes.  To  the  first 
add  a  few  drops  yoo^o'o  r^cm  solution.  To  the 
others  add  amounts  increasing  in  a  definite  ratio. 

At  first  there  will  be  no  effect  (immunity). 
As  the  amount  of  ricin  added  is  increased,  a  point 
will  be  reached  at  which  agglutination  of  red 
corpusles  will  be  produced.  This  is  the  neutrali- 
zation point. 

Evidently,  there  is  a  definite  quantitative 
chemical  relation  between  the  toxine  and  the 
antitoxine. 


IMMUNITY  71 

Theory  of  Immunity.1  —  Jenner  discovered  the 
protective  action  of  vaccinia  against  sniall-pox.  The 
small-pox  virus  when  passed  through  a  susceptible 
animal  becomes  attenuated.  This  weakened  poison 
introduced  into  the  circulation  in  man  protects  the 
individual  for  long  periods  against  the  original  disease 
—  it  establishes  an  artificial  immunity  against  small- 
pox. Schwann  found  that  fermentation  and  putre- 
faction arose  through  the  agency  of  micro-organisms 
coming  from  without.  Pasteur,  and  Koch  demonstrated 
that  the  inoculation  of  animals  with  pure  cultures  of 
certain  bacteria  produced  specific  infectious  diseases, 
and  that  these  cultures  could  be  modified  at  will, 
either  by  passing  through  the  animal  body,  as  in 
Jenner's  method,  or  in  artificial  culture  media.  Pas- 
teur produced  artificial  immunity  by  using  attenuated 
virus.  Behring  discovered  ^that  the  blood-serum  of 
animals  immunized  against  diphtheria  contained  a  sub- 
stance which  would  protect  other  animals  against  the 
toxine  of  diphtheria.  So  also  with  tetanus.  Ehrlich 
introduced  the  quantitative  study  of  toxiues  and  anti- 
toxines  by  means  of  test-tube  experiments,  thereby 
eliminating  the  uncertain  factor  of  the  animal  body. 
Thus  it  was  shown  in  experiments  on  tetanus  toxine 
that  the  action  of  antitoxines  is  accelerated  by  heat, 
retarded  by  cold,  dependent  on  concentration  —  in 
short,  that  it  is  a  chemical  action.  In  the  above'ex- 
periments    on   ricin,    it    is   shown    that    the    relation 

1  Ehrlich :  Croonian  Lecture,  Proceedings  of  the  Royal 
Society,  London,  1901,  lxvi,  pp.  424-118. 


72  FERMENTATION 

between  toxine  and  antitoxine  is  quantitative.  These 
results,  obtained  by  test-tube  experiments,  have  been 
confirmed  by  observations  on  living  animals.  Thus  it 
was  established  that  a  fixed  quantity  of  toxine  is  neu- 
tralized by  a  fixed  quantity  of  its  specific  antitoxine. 

Chemical  substances  atfect  only  those  tissues  with 
which  they  are  able  to  come  .  into  chemical  contact. 
They  must  first  reach  the  tissue.  This  general  law  is 
illustrated  by  the  experiments  of  Douitz  with  tetanus 
toxine.1  When  the  toxine  is  injected  directly  into 
the  circulation  and  immediately  followed  by  a  chemi- 
cally equivalent  amount  of  antitoxine,  the  animal  is 
not  poisoned  ;  all  the  toxine  circulating  in  the  blood 
is  neutralized.  When  the  same  neutralizing  dose  is 
injected  eight  minutes  after  the  toxine,  death  occurs 
from  tetanus  exactly  as  if  no  antitoxine  had  been  used. 
In  these  eight  minutes  a  lethal  quautity  of  toxine 
must  have  left  the  blood  and  entered  the  tissues. 
This  toxine  which  has  entered  the  tissues  may  still 
for  a  time  be  withdrawn  by  injection  of  the  specific 
antitoxine  in  quantities  much  greater  than  the  simple 
neutralizing  dose.  The  longer  the  delay,  the  larger 
the  saving  dose.  But  after  a  fixed  interval,  or  "period 
of  incubation,"  no  amount  of  antitoxine,  however 
large,  will  prevent  tetanus.  There  must,  therefore, 
be  present  in  the  brain  or  cord  (the  organ  princi- 
pally affected  by  tetanus  toxine)  certain  atom  groups 
which,  like  the  antitoxine,  have  a  chemical  affinity 
for  the  toxine.     At  the  close  of  the  period  of  incuba- 

1  Donitz  :  Klinisches  Jahrbuch,  1900,  vii. 


IMMUNITY  73 

tion  the  chemical  union  between  these  atom  groups 
and  the  toxine  is  complete  and  the  antitoxine  is  shut 
out.  Wassermann  l  found  that  when  tetanus  toxine 
was  mixed  with  fresh  brain  or  cord  substance  from  the 
guinea-pig,  the  toxine  united  chemically  with  the  nerve 
centres  so  that  neither  the  surrounding  liquid  nor  the 
mixture  itself  was  poisonous  when  injected  into  an 
animal. 

The  stable  benzene  ring  and  the  less  stable  side-chains 
of  the  benzene  derivatives  2  suggested  to  Ehrlich  that 
living  cells  also  consist  of  a  stable  centre  and  less  stable 
side-chains.  The  side-chains  enable  the  cell  to  form 
chemical  combinations  with  food  stuffs  and  other  bodies 
that  possess  atom  groups  having  a  chemical  affinity 
with  the  atom  groups  in  the  side-chains.  It  is  in  this 
way  that  the  toxine  is  bound  to"the  cell.  Experiments 
have  shown  that  the  binding  atoms  in  the  toxine 
molecule  are  not  the  poison  atoms.  If  for  a  portion 
of  fresh  toxine  there  be  determined  quantitatively  (1) 
the  killing  power  and  (2)  the  amount  of  antitoxine 
required  to  neutralize  the  toxine,  aud  if  the  remainder 
of  the  toxine  be  then  allowed  to  stand  for  a  time,  it 
will  be  found,  on  again  determining  the  toxic  power 
and  the  combining  power,  that  the  toxic  power  has  di- 
minished, while  the  combining  power  remains  almost 
the  same.  Hence,  two  separate  and  independent  groups 
exist.  Ehrlich  terms  the  combining  atoms  the  hapto- 
phore  group,  while  the  poison  atoms  are  the  toxophore 

1  Wassermann  :  Berliner  klinisclie  Wochenschrift,  1898. 

2  See  page  65. 


<4  FERMENTATION 

group.  The  haptophore  atom  group  (a-n-TU),  I  cling  to) 
unites  with  the  antitoxins,  if  there  be  any  present,  or 
with  any  other  atom  group  for  which  it  has  chemical 
affinity.  If  this  latter  atom  group  be  in  the  side-chain 
of  a  living  cell,  its  union  with  the  haptophore  atoms 
of  the  toxine  will  necessarily  bring  the  poison  atoms  of 
the  toxine  into  intimate  chemical  relationship  with  the 
central  atoms  of  the  cell.  Poisoning  will  then  take 
place.  If  the  cells  of  vital  organs  have  no  atom  groups 
with  chemical  affinity  for  the  haptophore  group  of  a 
toxine,  no  union  between  cell-atom  group  and  hapto- 
phore takes  place,  the  toxophore  is  not  brought  into 
intimate  contact  with  the  cell,  and  poisoning  does 
not  occur.  The  animal  is  naturally  immune  to  this 
particular  toxine.  Thus  a  toxine  in  sausages  is  exces- 
sively poisonous  to  ma*n,  the  monkey,  and  the  rabbit, 
while  even  large  amounts  are  not  injurious  to  the 
dog. 

The  haptophore  group  of  the  toxine  acts  immediately 
after  injection  into  the  organism,  while  in  most  or  all 
toxines  the  toxophore  group  becomes  active  only  after 
a  longer  or  shorter  incubation  period.  During  this 
period  the  animal  may  often  be  saved  by  placing  it  in 
conditions  in  which  the  toxophores  cannot  act.  Thus 
frogs  kept  at  less  than  20°  C.  are  not  poisoned  by  large 
doses  of  tetanus  toxine,  though  much  smaller  doses  are 
fatal  at  a  higher  temperature  (Morgenroth). 

The  toxophile  atom  group  of  the  cell  was  not  pre- 
destined to  unite  with  a  remotely  possible  toxine,  — 
it  has  a  normal  function,  probably  that  of  attaching 
food   to   the  cell.     When   it   enters,  into  its  firm  and 


IMMUNITY  75 

enduring  union  with  the  haptophore  group  of  a  toxine, 
this  normal  function  is  lost.  Such  a  loss  acts  as  a 
physiological  stimulus.1  Xew  side-chains  are  produced 
by  the  cell,  only  to  unite  with  fresh  toxine.  The  pro- 
duction and  the  loss  of  side-chains  continue  until  all 
the  toxine  in  the  blood  is  neutralized.  By  this  time 
the  cell  has  become  habituated  to  a  more  than  normal 
production  of  these  special  atom  groups.  The  excess 
is  cast  off  like  a  secretion  and  circulates  in  the  blood. 
These  free  side-chains,  possessing  a  special  affinity  for 
one  specific  toxine,  constitute  the  antitoxine  of  that 
toxine. 

Their  continued  production  after  the  neutralization 
of  all  the  toxine  protects  the  animal  against  fresh 
toxine,  i.  e.  establishes  continued  immunity. 

It  has  already  been  stated  that  by  special  means  the 
toxophore  group  of  a  toxine  may  be  weakened  or 
destroyed  while  its  haptophore  group  is  unchanged. 
Such  altered  and  non-poisonous  toxines  are  termed 
toxoids.  As  their  affinity  for  the  side-chains  of  the 
cells  remains  unaltered,  the  toxoids  by  continuing  to 
unite  with  the  side-chains  of  the  cells  may  stimulate 
the  production  of  such  side-chains  in  excess,  or,  in 
other  words,  may  assist  in  making  antitoxine  and  thus 
establishing  immunity. 

1  Weigerr :  Deutsche  medieinische  "Wochenschrift,  1896. 


76  FERMENTATION 


Haemolytic  and  Bacteriolytic  Ferments 

Bordet's  Experiments.1  —  Inject  into  the  perito- 
neum of  a  guinea-pig  10  c.c.  defibrinated  rabbit 
blood  on  five  successive  days.  After  two  more 
days  bleed  the  guinea-pig  and  obtain  the  serum, 
by  allowing  the  blood  to  stand  in  test-tubes  in  a 
cool  place  until  the  shrinking  clot  has  pressed 
out  the  serum. 

1.  Mix  a  drop  of  serum  from  a  fresh  guinea- 
pig  (one  not  injected  with  rabbit  blood)  with  a 
drop  of  defibrinated  rabbit  blood  and  examine 
under  the  microscope.  The  corpuscles  show  a 
very  slight  agglutination,  but  are  otherwise  un- 
injured. The  normal  serum  of  the  guinea-pig  is 
almost  inactive  upon  rabbit  blood. 

2.  A.  Mix  a  drop  of  the  serum  from  the 
injected  guinea-pig  with  a  drop  of  defibrinated 
rabbit  blood  and  examine  under  the  microscope. 
The  corpuscles  are  strongly  agglutinated.2 

B.  Mix  0.5  c.c.  of  the  serum  with  1.5  c.c. 
defibrinated  rabbit  blood. 

1  Bordet :  Amiales  de  1'Institut  Pasteur,  1898,  xii,  pp.  692- 
694. 

2  Agglutinated  blood  looks  granular,  especially  on  gentle 
shaking  ;  the  massed  corpuscles  sink  rapidly ;  they  will  not  pass 
through  filter  paper.  Agglutination  of  blood  corpuscles  is 
similar  to  the  clumping  of  the  typhoid  bacillus  in  the  serum  of 
a  typhoid-fever  patient. 


HAEMOLYTIC,    ETC.    FERMENTS  77 

The  corpuscles  are  agglutinated  and  their  hae- 
moglobin is  set  free.  The  mixture  becomes  red, 
clear  and  limpid  in  two  or  three  minutes.  With 
the  microscope  nothing  can  be  found  but  the 
stroma  of  the  corpuscles,  more  or  less  deformed, 
very  transparent  and  scarcely  visible. 

The  continued  presence  of  blood  corpuscles 
of  the  rabbit  in  the  blood  of  the  guinea-pig  has 
developed  in  the  latter  the  power  to  agglutinate 
the  corpuscles  and  to  set  free  their  haemoglobin. 
It  is  thus  that  the  guinea-pig  protects  itself ;  it 
acquires  immunity. 

3.  Heat  1  c.c.  of  serum  fco  55°  C.  for  half  an 
hour.  Add  0.5  c.c.  of  this  to  1 .5  c.c.  defibrinated 
rabbit  blood  as  in  Experiment  3. 

The  serum  which  was  heated  to  od°  C.  no 
longer  destroys  the  corpuscles,  but  still  strongly 
agglutinates  them.1 

Evidently  the  agglutination  of  the  corpuscles 
and  the  setting  free  of  the  haemoglobin  (termed 
"  hiking ")  are  effected  by  different  substances. 
The  agglutinating  body  resists  a  temperature  that 
destroys  the  blood-laking  body. 

4.  To  the  mixture  used  in  the  preceding  experi- 
ment, add  2  c.c.  of  fresh  serum  from  a  normal 

1  A  very  slow  destruction  of  the  red  corpuscles  may  be  ob- 
served. This,  however,  is  due  to  the  fresh  serum  in  the  1.5 c.c. 
defibrinated  rabbit  blood,  as  will  be  evident  from  Experiment  4. 


78  FERMENTATION 

guinea-pig  (one  that  has  not  been  iujected  with 
rabbit  blood). 

In  a  few  minutes  the  mixture  becomes  limpid 
and  red.     The  laking  power  is  restored. 

Obviously,  with  the  fresh  serum  was  added 
the  unstable  body  destructive  to  red  corpuscles.. 
Ehrlich  and  Morgenroth  have  shown  that  at  low 
temperatures  the  stable  body  unites  with  the  red 
corpuscles  while  the  unstable  body  remains  in 
the  serum ;  in  this  case  the  haemoglobin  is  not 
set  free.  At  higher  temperatures  the  haemoglo- 
bin separates  and  the  unstable  body  is  found  to 
have  left  the  serum.  It  has  joined  the  stable 
body  in  the  sediment. 

Following  the  side-chain  theory  already  men- 
tioned, Ehrlich  and  Morgenroth  assume  that  the 
stable  substance  has  two  combining  powers; 
on  the  one  hand  it  unites  with  the  red  corpus- 
cles, on  the  other  with  the  unstable  substance, 
thus  bringing  it  to  the  cell  which  it  may  then 
destroy. 

Immunity  against  toxines  and  foreign  red  cor- 
puscles are  only  two  of  the  protective  actions  of 
the  blood.  The  injection  of  cells  of  the  most 
varied  kinds  is  followed  by  the  production  of 
specific  protective  bodies ; *  thus,  the  injection  of 

1  Metchnikoff:  Annales  de  l'lnstitut  Pasteur,  1900,  xiv, 
p.  369. 


OXIDIZING   FERMENTS  79 

bacteria  causes  the  formation  of  bacteriolysines, 
which. destroy  the  injurious  organism. 

Many  haemolysines  and  agglutines  are  found 
in  plants  ;  others,  for  example,  the  tetanus  bacil- 
lus, are  bacterial ;  still  others,  such  as  snake 
venom,  are  animal  secretions. 

Oxidizing  Ferments 

Schonbein's  Experiment.1  —  1.  To  ten  grams 
hydrogen  peroxide  add  tincture  of  guiac  (freshly 
prepared  by  dissolving  guiac  resin  in  alcohol) 
drop  by  drop  until  the  liquid  is  milky.  Now 
add  from  eight  to  ten  drops  of  a  somewhat  con- 
centrated extract  of  malt,  prepared  in  the  cold. 

The  guiac  will  be  oxiclfzed  and  will  turn  blue. 

2.  Eepeat  the  experiment,  adding  in  place  of 
the  malt  extract  from  eight  to  ten  drops  of  blood. 

The  guiac  will  be  oxidized,  as  before. 

Further  Oxidations  by  Animal  Tissues.2  — 
1.  Soak  strips  of  bibulous  paper  in  a  diluted  solu- 
tion made  as  follows : 

a-naphthol 1  ruol. 

sodium  carbonate    ....     3     " 
para-phenylenediamine     .     .     1     " 

1  Schonbein  :  Zeitschrift  fiir  Biologie,  1868,  iv,  p.  367. 

2  Spitzer  :  Archiv  fiir  die  gesammte  Physiologie,  1895,  Ix, 
pp.  322-323. 


80  FERMENTATION 

This  solution,  left  in  the  atmosphere,  oxidizes 
slowly  to  indophenol  (violet  color). 

Place  a  drop  of  a  known  oxidizer,  e.g.  ferri- 
cyanide  of  potash  or  potassium  chroma te,  on  the 
saturated  paper. 

The  color  will  change  at  once,  in  consequence 
of  immediate  oxidation. 

(1)    C6H4(NH2)2  +  C10H7OH  +  0  = 

PARA-PHEXYLENEDIAMINE        A-NAPHTHOL  C«H.iXFTo 

nh<c:h6oh + w> 

&     KH/'H'NH!  +  O  -  N^C»H*NHa  +H  O 
*H<CwH6OH  +  °  -  ^<C„H.O      +H'° 


INDOPHENOL 

Each  of  the  combining  molecules  has  been  acted 
upon  by  a  different  oxygen  atom ;  hence  the  oxy- 
gen molecule  must  have  been  split. 

2.  Bub  the  test  paper  with  finely  divided  tissue 
from  the  liver  or  any  other  organ. 

Oxidation  will  occur.  B 

A  drop  of  blood  placed  on  the  test  paper  is 
soon  surrounded  by  a  characteristically  colored 
ring. 

Extraction  of  Nudeo-Proteicl  from  Liver}  — 
Perfuse  a  fresh  liver  (dog)  with  tap  water  until 
the   washings  are  no  longer  colored  bv  haemo- 


o 


1  Spitzer  :  Archiv  fur  die  gesammte  Physiologie,  1897,  lxvii, 
p.  616. 


OXIDIZING' FERMENTS  81 

globin.  Grind  the  liver  to  a  pulp  and  press 
through  several  thicknesses  of  gauze.  Add  five 
volumes  of  distilled  water.  Allow  the  mixture 
to  stand  twenty-four  hours  at  low  temperatures. 
Remove  the  opalescent  watery  extract  with  a 
pipette  and  filter  through  linen.  Demonstrate 
with  the  microscope  that  liver  cells  are  absent 
from  the  liquid.  Add  T7^  HC1  drop  by  drop  until 
there  is  no  further  precipitation,  and  the  super- 
natant fluid  is  clear.  Since  the  precipitate  redis- 
solves  in  acid,  use  lacmoid  as  an  indicator.  Cease 
when  the  lacmoid  shows  a  trace  of  excess.  De- 
cant the  precipitate,  filter,  wash  the  residue  with 
water. 

Oxidation  by  Nucleo-Proteid.  —  Place  in  a  wide- 
necked  flask  50  c.c.  waSer  containing  0.2  gram 
of  the  fresh,  brown  substance  and  10  c.c.  hydro- 
gen peroxide  in  a  small  glass  cup.  The  hydrogen 
peroxide  must  be  neutralized  with  from  1  to  1.5 
c.c.  y?o  XaOH.  Connect  the  flask  with  the  lower 
end  of  a  eudiometer  by  means  of  a  bent  tube. 
Shake  the  flask  so  that  the  hydrogen  peroxide 
shall  come  in  contact  with  the  tissue.  Oxygen 
is  at  once  set  free.  Read  in  the  eudiometer 
the  oxygen '  developed  from  minute  to  minute. 
Spitzer  found : 

After  minutes  .     .       12       3      4      5       8       9     16 

C.c.    02  developed    19    28    41    55    69    85    87    95 

6 


82  FERMENTATION 

Oxidation  about  the  Nucleus.1  —  Introduce  the 
oxidizable  solution  of  a-naphthol  and  para-phe- 
nylenediamine  (page  79),  beneath  the  cover 
glass  of  a  fresh  preparation  of  teased  thymus 
or  spleen. 

"  Granules  of  the  intense  greenish-blue  oxi- 
dation product  shortly  make  their  appearance 
within  the  leucocytes.  Their  first  appearance  is* 
typically  at  the  boundary  between  nucleus  and 
cytoplasm ;  eventually  the  latter  may  become 
so  densely  laden  as  completely  to  obscure  the 
nucleus.  .  .  .  The  nucleus  is  the  chief  agency  in 
the  intracellular  activation  of  oxygen.  The  ac- 
tive or  atomic  oxygen  is  in  general  most  abun- 
dantly freed  at  the  surface  of  contact  between 
nucleus  and  cytoplasm." 

Glycolysis  in  Blood.  Bernard's  Experiment? — - 
125  c.c.  dog's  blood  were  divided  into  five  equal 
parts.  The  sugar  in  each  was  estimated  as 
follows : 


Sugar 
Grams  per  1000 

1. 

Analysis 

made 

at  once      .     .     . 

.     1.07 

2. 

a 

a 

after  10  minutes 

.     1.01 

3. 

u 

a 

"     30        "      . 

.     0.88 

4. 

a 

a 

"       5  hours     . 

.     .     0.44 

5. 

a 

u 

"     24      " 

.     0.00 

1  Lillie  :  American  journal  of  physiology,  1902,  vii,  p.  420. 

2  Bernard  :  Comptes  rendus  de  I'acadetnie  des  sciences,  Paris, 
1876,  lxxxii,  p.  1406. 


OXIDIZING    FERMENTS  83 

Sugar  disappears  from  the  blood  on  stand- 
ing. 

It  has  been  found  by  Lepine  and  Barral *  that 
the  glycolytic  power  of  the  blood  increases  as  the 
temperature  rises  to  52.5°  C,  which  is  the  opti- 
mum.    At  54°  the  ferment  is  destroyed. 

Oxidation  not  Dependent  on  Living  Cells  of  Blood. 
—  Place  the  following  solutions  at  3-4-35°  C.  for 
six  hours,  allowing  a  stream  of  air  to  pass  through 
the  liquid.     Then  estimate  the  sugar.2 

A.    Calf's  blood 100  c.c. 

Water  containing  1.14  gram  grape  sugar       10  c.c. 
Seegen  3  recovered  1.000  gram. 

1  Lepine  and  Barral :  Comptes  rendus  de  Tacademie  des  sci- 
ences, Paris,  1891,  cxii,  p.  146.' 

2  Test  the  nitrate  by  adding  a  drop  of  acetic  acid  and  a  little 
ferrocyanide  of  potassium. 

The  absence  of  a  precipitate  shows  freedom  from  proteids  and 
ferric  salts.     Concentrate  filtrate  to  150-200  c.c. 

Titration  of  the  Sugar  Extract.  —  Make  the  volume  of  the 
solution  such  that  its  probable  content  of  sugar  shall  lie 
between  0.0004  and  0.0010.  Causse  (Bulletin  de  la  Societe 
chimique  de  Paris,  1,  p.  625)  recommends  that  1750  c.c.  of 
water  containing  5  grams  of  ferrocyanide  of  potassium  be  added 
to  each  250  c.c.  of  Fehling's  solution.  Boil  10  c.c.  of  this 
mixture  and  add  the  sugar  solution  drop  by  drop  until  the  blue 
liquid  is  decolorized  (Arthus :  Archives  de  physiologie,  1891, 
p.  425). 

3  Estimation  of  Sugar  in  Blood.  Extraction  of  the  Sugar 
from  the  Blood.  — To  350-400  c.c.  boiling  water  add  all  at  once 
50  c.c.  blood  containing  5  c.c.  one  per  cent  acetic  acid.     Let 


S-l  FERMENTATION 

B.    Calf's  blood 100  c.c. 

Water  containing  1.14  gram  grape  sugar  10  c.c. 

Chloroform 1  c.c. 

Seegen  recovered  0.9G0  grain. 

■    The  chloroform  destroys  the  cells,  but  fails  to 
check  the  oxidation. 

Relation  of  Glycolysis  to  the  Pancreas  and  the 
Lymph.1 — Remove  the  pancreas  aseptically  from  an 
anaesthetized  dog  which  has  fasted  thirty-six  hours. 
Estimate  the  sugar  in  the  urine  at  intervals  of  a  few. 
hours. 

Sugar  will  be  present  in  large  and  increasing  quanti- 
ties,2 rising  even  to  twenty  per  cent. 

Inject  into  the  jugular  vein  15-20  c.c.  of  lymph 
from  the  thoracic  duct  of  a  dog  fed  a  few  hours  before 
upon  one  litre  of  milk. 

the  mixture  boil  for  a  few  minutes.     Filter  through  a  small 
linen  cloth. 

Separation  of  Proteids.  —  Boil  the  filtrate.  Most  of  the  pro- 
teids  will  separate  by  coagulation.  The  remainder,  if  necessary, 
may  be  removed  by  adding  to  each  300  c.c.  of  filtrate,  5  c.c. 
saturated  solution  of  sodium  acetate,  and  a  small  quantity  of  a 
dilute  solution  of  ferric  chloride,  neutralizing  almost  completely 
with  dilute  soda  solution,  and  boiling.  The  ferric  chloride  will 
precipitate  as  ferrous  chloride  and  will  carry  down  the  last 
traces  of  proteid  substances.  Filter.  "Wash  with  boiling  water. 
(Seegen  :  Centra lblatt  fur  Physiol ogie,  1891,  v,  p.  824.) 

1  Lepine  :  Comptes  rendus  de  l'academie  des  sciences,  Paris, 
1890,  ex,  p.  742. 

2  Von  Mering  and  Minkowski :  Archiv  fiir  experimentelle 
Pathologie  und  Pharmakologie,  1890,  xxvi,  p.  371. 


OXIDIZING    FERMENTS  85 

The  glycosuria  will  greatly  diminish. 

After  a  few  hours,  the  glycosuria  will  become  once 
more  intense,  continuing  until  death.  The  quantity  of 
sugar  in  the  blood  is  also  greatly  increased. 

Glycolytic  Ferment  of  Pancreas.1  —  Eemove  the 
pancreas  aseptically  from  a  dog  immediately  after 
death.  Crush  it  at  once  in  100  c.c.  sterile  water 
containing- 0.2  gram  sulphuric  acid.  Allow  it  to 
macerate  two  hours  at  38°  C.  Neutralize  the 
acid  with  soda,  add  0.5  gram  pure  glucose,  and 
keep  the  mixture  one  hour  at  38°  C.  Estimate 
the  sugar. 

The  loss  will  be  from  ten  to  fifty  per  cent. 

When  pancreatic  extract  made  without  acid  is  used, 
the  loss  of  sugar  is  much  Jress.  Probably,  therefore, 
the  glycolytic  ferment  is  produced  from  a  zymogen  by 
hydration. 

Malt  diastase,  or  salivary  diastase,  kept  three  hours 
at  38°  in  water  containing  one  tenth  per  cent  sulphuric 
acid  loses  the  power  to  change  starch  to  sugar,  but  ac- 
quires a  glycolytic  power. 

If  the  pancreatic  juice  which  flows  upon  stimulation 
of  the  peripheral  end  of  the  vagus  (Pawlow)  is  treated 
with  dilute  acid,  1  :  1000,  the  amylolytic  power  is  lost, 
but  glycolytic  power  is  acquired.  During  the  excita- 
tion of  the  nerve  —  while  the  juice  is  flowing  —  the 

1  Lepine :  Comptes  rendus  de  l'academie  des  sciences,  Paris, 
1895,  cxx,  p.  139.' 


86  FERMENTATION 

blood  in  the  pancreatic  vein  has  almost  no  glycolytic 
power;  after  the  jnice  ceases  to  run,  the  blood  has 
considerable  glycolytic  power.  Here  the  external  is 
balanced  against  the  internal  secretion  of  the  pancreas. 

Oxidative  ferments  are  very  widely  distributed  both 
in  animals  and  plants.  The  above  experiments  show 
their  presence  in  the  blood,  pancreas,  liver,  and  lymph. 
They  are  present  also  in  the  urine. 

The  stomach  contains  a  ferment  that  oxidizes  lactone 
to  lactic  acid.1 

Urushi,  the  milky  secretion  of  Rhus  vernicifera,  dries 
in  the  air  to  a  translucent  varnish  (Japanese  lacquer). 
It  contains  urushic  acid,  which  does  not  dry  sponta- 
neously, and  a  ferment,  the  addition  of  which  to 
urushic  acid  causes  the  latter  to  dry  to  lacquer.  A 
sample  of  fresh  juice  boiled  to  stop  the  action  of  the 
ferment  on  urushic  acid  contained  15.01  per  cent 
oxygen  ;  lacquer  dried  in  the  usual  manner  contained 
20.52  per  cent  oxygen. 

Many  oxidations  are  effected  by  the  tissues  without 
the  aid  of  ferments,  so  far  as  is  yet  known.  These  be- 
long properly  to  metabolism,  but  in  passing,  it  may  be 
noted  that  while  substances  exceedingly  resistant  to 
oxidation,  for  example,  proteids,  are  oxidized  in  the 
body,  other  substances  very  easily  oxidizable  may  be 
excreted  unchanged ;  oxalic  acid  is  one  of  these.2 

1  Haramarsten  :  Maly's  Jahresbericht  derThierchemie,  1872, 
ii,  p.  118.       (Original  in  Swedish.) 

2  Pohl:  Arehiv  fur  experhnentelle  Pathologie  und  Pharraa- 
kologie,  1896,  xxxvii,  p.  413. 


ALCOHOLIC    FERMENTATION  87 

Hoppe-Seylers  Theory.1  —  Living  tissues  consist  of 
easily  combustible  reducing  substances,  which  split 
the  oxygen  molecules,  taking  to  themselves  one  atom 
of  0  and  setting  the  other  free  in  active  state  to 
unite  with  any  oxidizable  substance  present. 

Traube's  Theory? — Iu  living  protoplasm  oxygen  is 
rendered  active  by  an  oxidizing  ferment,  which  brings 
the  oxygen  to  bodies  ordinarily  oxidizable  only  by  such 
powerful  agents  as  heat  and  strong  alkalies. 

Inorganic  bodies,  e.  g.  platinum  black,  the  oxides  of 
copper,  silver,  mercury,  and  vanadium,  and  certain  iron 
salts  similarly  act  as  oxygen  carriers.      Thus 

(1)  Pt  -f  0,  +  H,  =  PtO  +  H20 

(2)  PtO  +  H2  =  Pt  +  H20 

The  oxygen  carrier  reduces  H202,  takes  one  atom  0 
to  itself,  then  gives  off  this  atom  in  an  active  or 
nascent  state  to  oxidize  #hy  oxidizable  compound 
present ;  e.  g.  guiac  Grape  sugar  takes  0  from 
indigo-blue,  producing  thereby  indigo-white.  The 
indigo-white  oxidizes  itself  to  indigo-blue,  then  gives 
up  another  atom  of  0,  and  so  on. 

Alcoholic  Fermentation 
The  Yeast  Plant.  —  Observe  a  solution  of  sugar 
undergoing    alcoholic    fermentation.3      Xote    the 
bubbles  of  gas,  the  scum,  the  sour  odor. 

1  Hoppe-Seyler:  Zeitsehrift  fur  physiologische  Chemie,  1879, 
ii,  p.  1. 

2  Traube  :  Berichte  der  deutscben  chemischen  Gesellschaft, 
1883,  xvi,  pp.  123, 1201,  and  earlier  papers  in  volumes  x  and  xv. 

3  The  fermentation  is  assisted  by  providing  the  yeast  plant 


88  FERMENTATION 

Examine  some  of  the  mixture  under  the  micro- 
scope. Note  the  multitude  of  globular  or  slightly 
ovoid  bodies,  the  largest  about  T^  mm.  in  diame- 
ter. They  are  motionless.  Many  have  put  forth 
buds.     They  seem  to  be  plants  in  active  growth.1 

1.  Place  300  c.c.  of  the  nutrient  liquid  (Ex- 
periment 1)  in  a  flask  holding  500  c.c.  Add  a 
piece  of  fresh  compressed  yeast  the  size  of  a  pea. 
Place  the  flask  in  a  temperature  of  35°  C. 

Note  that  as  fermentation  advances  the  yeast 
increases  in  quantity. 

2.  Place  a  small  piece  of  fresh  compressed 
yeast  in  a  test-tube.  Fill  the  tube  with  nutrient 
liquid  and  invert  it  in  a  dish  of  similar  liquid.  The 
tube  may  be  kept  upright  by  a  clamp.  Let  the 
mixture  stand  twenty -four  hours  in  a  warm  room. 

with  the  salts  present  in  the  ash  of  yeast  (Pasteur).     A  useful 
substitute  is 

Potassium  phosphate     ...  20  gms. 

Calcium  phosphate        ...  2 

Magnesium  sulphate      ...  2 

Ammonium  tartrate       .     .     .         100 

Cane  sugar 1,500 

Water 8,376 

10,000 
(Practical  biology,  Huxley  and  Martin.) 
1  Cagniard-Latour  :  L'Institut,  1835,  iii,  p.  150  ;  also  Annates 
de  chimie  et  de  physique,  1838,  lxviii,  p.  206.  The  yeast  plant 
was  first  observed  microscopically  in  beer-yeast  by  Leeuwen- 
hoek,  1680,  but  he  did  not  associate  fermentation  with  the 
growth  of  the  yeast. 


ALCOHOLIC   FERMENTATION  89 

The  tube  will  fill  with  gas.  "With  a  bent  pipette 
introduce  about  1  c.c.  of  a  solution  of  sodium 
hydroxide  (sp.  gr.  1.12  =  11  per  cent).  The  gas 
will  be  absorbed,  with  formation  of  sodic  carbon- 
ate, and  the  liquid  will  rise  in  the  tube. 

The  growth  of  the  yeast  plant  is  accompanied 
by  the  production  of  carbon  dioxide. 

3.  Return  to  Experiment  1.  After  the  fer- 
menting liquid  has  ceased  to  give  off  gas,  place  a 
stopper  with  a.  bent  tube  in  the  mouth  of  the 
flask  and  -distill  the  contents  of  the  flask  in  a 
water  bath.  Condense  the  first  fifth  of  the  dis- 
tillate. Saturate  this  with  sodium  carbonate. 
Redistill,  and  condense. 

Test  for  alcohol  by  warming  the  distillate  with 
potassium  dichromate  and  dilute  sulphuric  acid, 
whereby  the  alcohol  will  be  oxidized  to  aldehyde, 
with  characteristic  odor. 

Alcohol  is  present. 

The  production  of  alcohol  by  the  yeast  is  the  work 
of  the  ferment  zymase.1  This  body  is  closely  bound 
to  the  protoplasm  of  the  cell,  very  easily  destroyed,  not 
produced  in  excess,  and  not  secreted  free.  Only  sugars 
containing  three,  six,  and  nine  carbon  atoms  are  at- 
tacked. The  saccharobioses  must  be  "  inverted"  be- 
fore they  can  be  fermented.     Thus,  cane  sugar  must 

1  Buclmer  :  Berichte  der  deutschen  chenrisclien  Geaellschaft, 
1897,  xxx,  pp.  117,  1110,  2668. 


90  FERMENTATION 

first  be  inverted  to  grape  sugar  by  invertin,1  and 
malt  sugar  by  maltase.  Lactase  is  present  in  some 
yeasts,  enabling  them  to  ferment  milk  sugar.  Diastase 
is  also  found. 

The  action  of  these  several  ferments  becomes  clear 
when  the  chemical  nature  of  the  carbohydrates  is 
recalled.   * 

Chemical  Relations  of  Carbohydrates.  —  Carbohy- 
drates were  formerly  defined  to  be  compounds  con- 
taining six,  or  a  multiple  of  six  carbon  atoms,  together 
with  hydrogen  and  oxygen  atoms  in  the  proportion  in 
which  they  exist  in  water.  The  researches  of  E. 
Fischer  have  shown  that  all  aldehydes  (bodies  which 
are  the  first  oxidation  products  of  primary  alcohols,  and 
which  contain  the  carbouyl  group  CO)  and  all  ketones 
(bodies  which  are  the  first  oxidation  products  of  second- 
ary alcohols  and  which  likewise  contain  the  carbouyl 
group  CO)  contain  carbon,  hydrogen,  and  oxygen,  there 
being  two  atoms  of  hydrogen  to  one  atom  of  oxygen, 
as  in  water. 

The  carbohydrates,  therefore,  no  longer  occupy  an 
isolated  position,  but  are  to  be  classed  with  the  fats, 
being  metliane  derivatives  in  which  the  carbon  atoms 
are  arranged  in  an  open  chain;  thus,  grape  sugar  is  an 
aldehyde  alcohol,  and  fruit  sugar  a  ketone  alcohol. 

The  carbohydrates  are  divided,  according  to  the  size 
of  their  molecule,  into  monosaccharides,  disaccharides, 
and  polysaccharides.     The  monosaccharides  (e.  g.  grape 

1  For  extraction,  see  Lea  :  Journal  of  physiology,  1885,  vi, 
p.  142. 


ALCOHOLIC    FERMENTATION 


91 


sugar)  are  the  first  oxidation  products  of  the  hexahy- 
dric  alcohols;  the  higher  carbohydrates  are  anhydrides 
of  the  monosaccharides.  Most  of  the  higher  carbohy- 
drates cannot  be  fermented  directly,  but  must  first 
be  hydrolyzed  (i.  e.  take  up  water).  This  hydrolysis 
may  be  accomplished  by  the  prolonged  action  of  dilute 
acids  at  high  temperatures,  by  the  action  of  water  at 
still  higher  temperatures,  or  by  specific  ferments,  e.  g. 
diastase,  at  the  relatively  low  temperature  of  the  body. 
The  polysaccharides,  consisting  of  the  starches,  the 
gums  (e.  g.  dextrine  or  starch  gum)  and  the  celluloses 
(wood  fibre)  differ  greatly  from  the  lower  carbohy- 
drates. The  polysaccharides  are  usually  amorphous 
and  are  not  easily  soluble  in  water. 


Carbohydrates.1 


Glucoses,  Monoses 
CeH^Oe. 

Saccharobioses 

C12H22°11- 

Polysaccharides 
(C6H10O5)x. 

Grape  sugar — 
Grape  sugar — 

-Malt  sugar — 

—Starch 

Grape  sugar — 
Fruit  sugar — 

-Cane  sugar 

Grape  sugar — 
Galactose — 

-Milk  sugar 

1  Richter's  Organic  Chemistry,  Third  American  Edition,  i, 
p.  121. 


92 


FERMENTATION 


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FERMENTATION  93 


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94  BLOOD 

The  zymase  attacks  only  those  sugars  which  present 
a  specific  stereo-contiguratiun.  The  position  of  their 
atoms  in  space  must  fit  the  position  of  the  atoms  of 
the  ferment  (the  lock  and  the  key).  Thus,  only  the 
dextro-rotatory  forms  of  the  aldehyde  sugars  (d-glu- 
cose,  d-mannose,  d-galactose)  are  attacked  ;  the  sugars 
that  rotate  the  plane  of  polarized  light  to  the  left 
are  not  attacked.  It  is  probable  that  the  zymase 
of  different  species  of  yeast  presents  characteristic  dif- 
ferences. It  is  known  that  the  products  formed  in  the 
fermentation  of  sugar  by  different  species  of  yeasts  are 
to  a  large  degree  characteristic.  Often  these  products 
are  injurious.  Upon  this  specific  action  of  ferments 
testa  the  work  of  Hansen,1  who  taught  the  brewers  to 
make  pure  cultures  of  the  most  favorable  species  of 
yeast,  and  thereby  raised  the-  brewing  industry  to  the 
level  of  an  applied  science. 


BLOOD 
Specific  Gravity 

Drawing  the  Blood.  —  Wash  the  lobe  of  the  ear 
with  a  bit  of  absorbent  cotton  dipped  in  clean 
water.2  Bub  the  lobe  dry  with  another  piece 
of    cotton.     Pass    a  three-sided    surgical   needle 

1  Hansen:  Untersuelmngen  an  der  Praxis  der  Gahrungs- 
Industrie,  1895. 

2  Subjects  who  are  "bleeders"  are  not  to  be  used  for  this 
observation. 


SPECIFIC    GRAVITY  95 

through  a  Bunsen  flame.  (Do  not  heat  the 
needle  red  or  the  temper  will  be  drawn  and  the 
sharpness  lost.)  Stretch  the  skin  of  the  lobe 
between  the  fingers  of  the  left  hand.  Make  a 
quick  puncture  one-eighth  inch  deep  in  the  edge 
of  the  lobe.  Press  gently  to  start  the  flaw; 
The  blood  must  now  flow  freely.  On  no  account 
use  blood  squeezed  out. 

Determination  of  Specific  Gravity.1  —  Fill  a 
small  beaker  half  full  of  a  mixture  of  benzol  and 
chloroform  of  a  specific  gravity  of  about  1059. 
Let  a  drop  of  the  blood  fall  into  this  mixture. 
The  drop  will  remain  spherical,  for  blood  does 
not  mix  with  benzol  and  chloroform.  If  the 
drop  sinks,  add  chloroform  drop  by  drop,  mean- 
while stirring  the  mixture  with  a  glass  rod,  until 
the  drop  neither  rises  to  the  surface  nor  sinks 
to  the  bottom  but  swims  with  the  mixture.  If 
the  drop  rests  upon  the  surface,  add  benzol  in 
a  similar  manner.  When  the  drop  neither  sinks 
nor  floats,  its  specific  gravity  must  be  that  of  the 
benzol-chloroform  mixture.  Pour  the  mixture 
into  a  glass  cylinder,  through  a  piece  of  linen  to 
hold  back  the  blood-drop,  and  take  the  specific 
gravity  of  the^benzol-chloroform  with  an  areoni- 

1  Roy  :  Journal  of  physiology,  1884,  v,  p.  ix. 
Hammerschlag,  A.  :  Wiener  klinische  Wochenschrift,  1890, 
iii,  p.  1018. 


96  BLOOD 

eter.  The  result  is  also  the  specific  gravity  of 
the  blood. 

The  values  obtained  are  slightly  too  low.  The 
error  is  one  unit  in  the  third  decimal  place. 

Determine  the  specific  gravity  of  the  blood 
under  the  following  conditions.  Record  the  re- 
sults in  the  laboratory  note-book.  Hand  to  the 
instructor  a  copy  of  your  observations  written  in 
ink  upon  a  laboratory  blank.  The  material  col- 
lected by  the  class  will  be  analyzed  statistically 
by  a  committee  and  a  report  made. 

1.  The  specific  gravity  of  the  blood  in  a  healthy 
man. 

2.  In  the  same  man  half  an  hour  after  drink- 
ing 750  c.c.  of  water. 

3.  In  the  same  man  one  hour  after  drinking 
750  c.c.  of  water. 

4.  In  the  same  man  after  profuse  sweating. 
Note  any  feeling  of  thirst. 

5.  In  a  healthy  woman. 

Hammerschlag  found  the  specific  gravity  in 
chlorosis  and  nephritis  diminished  as  the  haemo- 
globin diminished.  Xo  relation  was  observed 
between  the  appearance  of  oedema  and  a  reduc- 
tion in  the  specific  gravity. 

Counting  the  Red  Corpuscles.  —  See  that  the 
pipettes  of  the  Thoma-Zeiss  apparatus  are  per- 
fectly clean  and  dry.     Open  the  bottle  contain- 


SPECIFIC    GRAVITY  97 

ing  Gower's  solution  (sodium  sulphate,  7.3  grams  ; 
acetic  acid,  20  c.c;  water,  125  c.c).  Prick  the 
ear  as  directed  on  page  94.  In  a  large  drop 
which  has  collected  without  pressure  put  the 
point  of  the  smaller  Thoma-Zeiss  pipette  ("red 
counter").  Fill  the  pipette  to  the  mark  0.5  by 
careful  suction.  Should  the  mark  be  passed, 
lower  the  column  to  the  mark  by  touching  the 
point  of  the  pipette  to  filter  paper.  When  the 
mark  is  reached,  clean  the  outside  of  the  pipette, 
dip  the  end  in  Gower's  diluent  solution,  and 
draw  the  liquid  very  carefully  up  to  the  mark 
101.  (Should  the  liquid  pass  the  mark,  the 
pipette  must  be  cleaned  and  dried  and  the  whole 
process  repeated.)  Close,  the  ends  of  the  pipette 
with  the  ringers,  and  shake  it  gently  for  one 
minute  in  order  to  mix  the  blood  thoroughly 
with  the  diluent.  The  blood  will  now  be  diluted 
200  times  its  volume. 

Remove  the  rubber  tube  from  the  pipette. 
Blow  out  the  unmixed  solution  in  the  capillary 
tube,  between  the  point  and  the  bulb,  and  several 
drops  of  the  mixture  in  the  bulb.  Wipe  off  the 
end  of  the  pipette.  Touch  it  to  the  ruled  disc. 
Let  a  very  small  drop  flow  out.  Place  the  cover- 
glass  on  the  drop.  The  flattened  drop  should 
almost  cover  the  glass.  If  it  spread  into  the 
moat,  clean  the  disc  and  use  a  second,  smaller 
7 


98  BLOOD 

drop.  Tf  Newton's  color-rings  cannot  be  seen 
between  the  coverglass  and  the  disc  by  placing 
the  eyes  near  the  level  of  the  coverglass,  another 
preparation  must  be  made,  with  cleaner  disc  and 
coverglass. 

Use  Leitz  No.  5  or  Zeiss  D  objective.  Bring 
the  drop  into  focus  and  then,  using  the  microm- 
eter screw,  find  the  ruled  field. 

On  the  central  portion  of  the  disc  1  square 
millimetre  has  been  ruled  into  400  squares,  each 
square  having  therefore  an  area  of  ^^  square 
millimetre.  Each  16  small  squares  are  sur- 
rounded by  double  lines,  thus  forming  a  "  large 
square."  In  the  Zappert-Ewing  slide,  the  cen- 
tral square  of  1  mm.  is  surrounded  by  eight  other 
squares  of  1  mm.  each,  and  the  central  ruling  is 
extended  through  the  surrounding  squares,  which 
are  intersected  by  lines  ^  mm.  apart.  Count  the 
number  of  corpuscles,  square  by  square,  in  200 
small  squares.  Corpuscles  touching  the  north 
and  south  lines  of  each  area  are  to  be  counted 
in,  those  touching  the  east  and  west  lines  are  to 
be  omitted  from  the  count. 

Each  square  has  an  area  of  ^J^  square  milli- 
metre. The  thickness  of  the  layer  of  blood,  i.  e. 
the  distance  from  the  ruled  disc  to  the  cover- 
glass,  is  0.1  mm.  The  volume  of  the  space  above 
each  square,  therefore,  is  ^J^  cubic  millimetre. 


SPECIFIC    GRAVITY  99 

As  the  blood  is  diluted  200  times  its  volume,  and 
the  number  of  squares  counted  is  200,  the  total 
number   of   corpuscles   in  a  cubic   millimetre  is 

x  x  200  x  4000 
200 

x  being  the  total  number  of  corpuscles  counted. 
In  short,  to  obtain  the  number  of  corpuscles  in  a 
cubic  millimetre,  multiply  by  4000  the  number 
counted  in  200  squares.  Clean  the  pipette  as 
soon  as  the  counting  is  done. 

Cleaning  the  Pipette.  —  Draw  clean  Gower's 
solution  through  the  pipette,  then  alcohol,  and 
finally  ether.  Dry  the  pipette  by  sucking  (not 
blowing)  air  through  \tM 

Control  Counting.  —  Count  the  red  corpuscles 
in  a  second  drop.  If  the  result  differ  greatly 
from  that  of  the  first  count,  the  corpuscles  in  a 
third  drop  must  be  counted. 

Counting  the  White  Corpuscles.  —  Have  ready 
a  diluting  solution  of  glacial  acetic  acid  (one- 
third  of  one  per  cent).  This  solution  will  make 
the  red  cells  invisible.  Obtain  a  very  large  drop 
of  blood.  Fill  the  large  Thoma-Zeiss  pipette 
(white    counter)  by  very  gentle  suction.     Keep 

1  Pipettes  left  dirty  will  be  cleaned  at  the  student's  ex- 
pense, or,  where  necessary,  a  new  one  purchased.  The  cost  is 
considerable. 


100  BLCOD 

the  pipette  nearly  horizontal,  both  in  obtain- 
ing the  drop  and  in  drawing  in  the  diluting  solu- 
tion ;  the  bottle  should  be  tilted.  Count  the 
white  corpuscles  in  the  entire  ruled  disc.  Eepeat 
with  a  second,  drop. 

Estimation  of  Haemoglobin 

Hoppe-Seyler's  Method1  (modified).  —  "Weigh 
about  five  grams  of  crystallized  haemoglobin. 
Make  a  concentrated  solution  in  a  measured 
quantity  of  distilled  water.  Saturate  with  car- 
bonic oxide.  Preserve  in  glass  tubes  containing 
about  6  c.c.,  the  ends  drawn  out  and  closed  in 
the  Bunsen  flame.  Before  using,  dilute  with 
distilled  water  to  0.2  per  cent  CO-Haemoglobin 
and  saturate  with  CO.  Place  in  one  compart- 
ment of  the  double  container  with  parallel  glass 
sides.  In  the  other  compartment  place  about 
2  c.c.  of  0.1  per  cent  sodium  hydrate  solution. 
Prom  a  drop  of  blood  obtained  from  the  ear  as 
directed  on  page  94  fill  the  pipette  to  the  mark 
(3  cubic  millimetres;  by  careful  suction.  Should 
the  mark  be  passed,  lower  the  column  to  it  by 
touching  the  point  of  the  pipette  to  filter  paper. 
When  the  mark  is  reached,  clean  the  outside  of  the 
pipette,  and  then  blow  the  contents  of  the  pipette 

1  Hoppe-Seyler  :  Handbuch  der  physiologisch-  una  patholo- 
gisch-chemischen  Analyse,  1893,  p.  412. 


HAEMORRHAGE   AND   REGENERATION  101 

into  the  0.1  per  cent  sodium  hydrate  solution. 
Eemove  all  the  blood  by  drawing  the  sodium 
hydrate  solution  in  and  out  of  the  pipette.  Look 
at  the  parallel  columns  of  haemoglobin  solution 
and  blood  solution  through  the  blackened  tube 
against  an  evenly  illuminated  sheet  of  white 
paper  plaeed  ten  inches  away.  Dilute  the  blood 
with  0.1  per  cent  XaOH  solution  until  its  color 
is  precisely  that  of  the  haemoglobin  solution. 

Measure  the  volume  of  the  two  solutions  in 
c.c.  Then  the  volume  of  the  haemoglobin  («) 
solution  is  to  the  known  quantity  of  haemo- 
globin which  it  contains  («')  as  the  volume 
of  the  blood  solution  (b)  is  to  the  desired  weight 
of  haemoglobin  (x)  it  contains,  or  the  weight  of 
haemoglobin  in  three  cubic  millimetres  of  the 
blood. 

Haemorrhage  and  Regeneration 

Determine  the  specific  gravity,  number  of  red 
and  white  corpuscles  per  millimetre,  and  per- 
centage of  haemoglobin  in  the  same  animal  under 
the  following  conditions :  Xormal ;  two  hours 
after  a  profuse  haemorrhage ;  one  day,  three  days, 
and  five  days  after  the  haemorrhage.  Plot  all 
three  curves  upon  one  co-ordinate  system. 


102  BLOOD 


Alkalinity 


Zuntz-Loewy  1-Eiigel 2  Method.  —  Place  j^  tar- 
taric acid3  in  a  finely  graduated  pipette  (1  c.c. 
in  20)  with  glass  stopcock. 

Place  in  a  small  beaker  5  c.c.  of  water  dis- 
tilled in  glass  and  proved  neutral  in  reaction. 
Select  the  larger  pipette  of  the  Thoma-Zeiss 
haemocytometer.  Note  the  cubic  millimetres 
contained  in  the  pipette4  up  to  the  mark  0.5. 
Remove  this  quantity  from  the  water.  Dry  the 
pipette  with  alcohol  (page  99).  Fill  the  pipette 
to  the  mark  0.5  with  blood  from  the  ear.  Re- 
move any  blood  from  the  outside  of  the  tube. 
Place  the  end  of  the  tube  in  the  neutral  water 
and  gently  expel  the  blood.  Wash  out  the  last 
traces  of  blood  by  gently  drawing  the  water  in 
and  out  of  the  tube.  If  this  be  done  with  care, 
the  mixture  will  now  measure  practically  5.0 
c.c.  including  the  blood.  Titrate  the  mixture 
with  the  Y5  tartaric  acid  solution  drop  by  drop. 

1  Loewy  :  Archiv  fiir  die  gesammte  Physiologie,  1894,  lviii, 
p.  462. 

2  Engel  :  Berliner  klinische  Wochenschrift,  1898,  xxxv, 
p.  308. 

3  A   normal   solution   of  tartaric   acid    (C4Hc06)    contains 

-^J —  75  g.    in  1  litre.     A   ^  tartaric  acid  solution 

contains  ff '=  1  g.  in  1  litre. 

4  This  must  be  determined  for  each  pipette. 


ALKALINITY  103 

Before  and  after  the  addition  of  each  drop  of 
the  acid  let  a  drop  of  the  mixture  fall  gently 
on  a  piece  of  light-colored  glazed  litmus  paper 
previously  impregnated  with  saturated  neutral 
sodium  chloride  solution.  In  such  paper  the 
salts  on  which  the  alkaline  reaction  of  the 
blood  largely  depends  diffuse  more  rapidly  than 
the  haemoglobin.  The  latter  forms  a  yellow  spot 
which  is  surrounded  by  a  clear  solution  of  the 
blood  salts.  When  the  titration  acid  has  com- 
bined with  all  the  alkali  in  the  blood  a  sharp 
red  ring  will  appear  around  the  yellow  spot  of 
haemoglobin.  This  ring  is  clearer  when  the 
blood  is  wiped  off  after  having  rested  a  few 
moments  on  the  paper.**" 

Normally,  the  alkalinity  of  0.05  c.c.  blood  is 
satisfied  by  about  5  c.c.  y\  tartaric  acid.  Then 
100  c.c.  blood  would  require  0.5x20x100  = 
1000  c.c.  y\  tartaric  acid.  1000  c.c.  =  l  litre 
normal  tartaric  acid  saturates  40  g.  XaOH.  1 
litre  y\  tartaric  acid  =  -f  £  =533  mg.  NaOH.  The 
alkalinity  of  100  c.c.  blood  =  533  mg.  XaOH.2 
Hence  0.05  c.c.  blood  =  0.25  mg.  XaOH. 

1  The  same  tint  of  paper  should  he  used  in  'comparative 
experiments.  Light  colored  papers  should  be  observed  by  di- 
rect light ;  darker  papers  by  transmitted  light.  Litmus  may 
be  replaced  by  lacmoid  (for  directions,  see  Cohnstein  :  Virchow's 
Archiv  fur  allgemeine  Pathologie,  cxxx,  and  Bockmann  :  Che- 
misch-technische  L'ntersuchungs-Methoden). 

2  The  average  given  by  Engel  is  426.4  —  533.0  mg.  XaOH. 


104  kespiration 

Coagulation  Time. 

Fasten  on  a  glass  slide  a  metal  ring  pierced 
with  a  small  hole.  Place  a  little  vaseline  on 
the  upper  surface  of  the  ring.  Upon  this  lay  a 
perfectly  clean  cover  glass  upon  the  middle  of 
the  under  surface  of  which  has  been  placed  a 
large  drop  of  blood  drawn  from  the  ear  with  the 
precautions  mentioned  on  page  94  As  soon  as 
the  drop  is  in  place  within  the  cell,  make  it  ro- 
tate by  blowing  gently  against  it  by  means  of  a 
pointed  glass  tube  applied  to  the  hole  in  the  metal 
ring. 

The  drop  will  cease  to  move  when  coagulation 
sets  in. 

Note  the  interval  between  the  drawing  of  the 
blood  and  the  onset  of  coagulation. 

The  method  is  rough,  and  a  fairly  correct  re- 
sult requires  much  care  and  a  number  of  obser- 
vations, but  even  thus  it  reveals  the  important 
diminution  in  coagulability  in  certain  diseases, 
e.  g.  jaundice. 

RESPIRATION 

Chemistry    of    Eespiration 

Estimation  of  Oxygen,  Carbon  Dioxide,  and 
Water.1  —  Weigh  bottles    3,  4,  and  5  (4  and  5 

1  Apparatus.  — Two  aspirator  bottles,  with  box.  A  wooden 
tray,  containing  a  jar  for  the  guinea-pig,  and  six  bottles,  viz. : 


CHEMISTRY   OF   RESPIRATION 


105 


together).  Place  the  guinea-pig  in  the  jar  and 
weigh.  During  one  hour  draw  air  through  bot- 
tles 1  to  6  by  placing  an  aspirator  bottle  on  its 
box  and  allowing  the  water  to  flow  from  this 
bottle  to  the  one  remaining  on  the  desk.  The 
rubber  connecting  tube  must  be  changed  when 
the  aspirator  bottles  are  changed.  After  one 
hour  weigh  bottle  3,  and  bottles  -i  and  5. 
Tabulate  results  as  follows  : 


Grams. 


Weight  of  jar  and  guinea-pig  at  beginning 
"                "                "              end     . 
Loss 

Wi.  of  bottle  3  (sulph.  acid)  at  beginning 
"  t         end     . 
Gain  (=  water  absorbed)  .     .     . 

Weight  of  bottles  4  and  5  at  beginning 
"  "  "         end    .     . 

Gain  (=  carbon  dioxide  absorbed) 

Total  water  and  carbon  dioxide  absorbed 
Loss  in  weight  of  jar  and  guinea-pig     . 
Difference  (=  oxygen  absorbed) 
Respiratory  quotient 


^Tos.  1  and  4,  filled  with  soda-lime,  to  absorb  carbonic  acid  ; 
Kos.  2,  3,  and  5,  filled  with  pumice  stone  soaked  in  sulphuric 
acid,  to  absorb  moisture  ;  Xo.  6,  a  Miiller  valve,  to  prevent  air 
being  forced  back  through  the  series  of  bottles  by  a  wrong 
coupling  of  the  aspirator  tubes. 


106  respiration 

Mechanics  of  Eespiration 

Artificial  Scheme.  —  Eaise  the  left  glass"  rod 
above  the  opening  in  the  rubber  tubing.  Hold 
the  lower  end  of  the  free  cylinder  even  with  the 
rubber  balloon,  and  pour  in  water  till  the  level 
just  reaches  the  balloon.  Lower  the  left  glass 
rod  to  cover  the  opening. 

The  surface  of  the  water  in  the  attached 
cylinder  represents  the  diaphragm  and  movable 
chest-walls;  the  interior  of  the  cylinder  above 
the  water,  the  thoracic  cavity ;  and  the  rubber 
balloon,  the  lungs.  The  left  manometer  shows 
the  intra-thoracic  pressure  ;  the  right  manometer 
shows  the  intra-pulmonary  pressure.  The  left 
glass  rod  closes  the  entrance  to  the  cylinder, 
i.  e.  makes  the  thoracic  cavity  a  closed  cavity, 
as  is  normal ;  the  right  glass  rod,  with  its  lower 
end  partly  covering  the  opening  in  the  rubber 
tubing,  controls  the  entrance  to  the  balloon  (the 
respiratory  passages). 

Inspiration.  —  Nearly  close  the  respiratory  pas- 
sage. Lower  the  water  level  to  the  base  of  the 
thoracic  cylinder. 

Note  the  change  in  the  size  of  the  lung,  and 
in  the  pressure  in  the  lung  and  in  the  thorax. 
Give  reasons  for  these  changes. 

Expiration.  —  Widen   the   respiratory   passage 


MECHANICS    OF   RESPIRATION  107 

slightly.  Eaise  the  water  level  slowly  till  the 
lung  is  slightly  but  evenly  distended. 

Note  the  pressure  in  the  pleural  cavity.  Is  it 
positive  or  negative  ?     Why  ? 

Normal  Respiration.  —  Slowly  and  rhythmi- 
cally raise  and  lower  the  diaphragm  (water  level) 
between  the  inspiratory  and  expiratory  level, 
taking  care  that  the  lung  never  becomes  even 
slightly  collapsed  at  the  end  of  expiration. 

Give  reasons  for  the  changes  in  the  intra- 
pulmonary  pressure. 

Forced  Respiration.  —  Eaise  and  lower  the 
diaphragm  more  quickly. 

Observe  that  the  differences  in  pressure  are 
increased.  *- 

Obstructed  Air  Passages. — Diminish  the  inlet 
in  the  respiratory  tube  by  moving  the  glass  plug. 
Eaise  and  lower  the  diaphragm. 

The  differences  of  pressure  will  be  increased. 

Asphyxia.  —  Close  the  entrance  to  the  lungs 
entirely. 

Note  the  effect  of  movements  of  the  diaphragm 
upon  the  intra-thoracic  and  intra-pulmonary 
pressures. 

Coughing  :  Sneezing.  —  Eemove  the  glass  rod 
from  the  respiratory  passage.  Bring  the  lung  to 
full  inspiration.  Close  the  respiratory  opening 
with  the  moistened  thumb.     Eaise  the  diaphragm 


108  RESPIRATION 

half-way  toward  expiration.  Suddenly  open  the 
respiratory  passage. 

Air  is  quickly  and  forcibly  expelled  from  the 
lung  (cough,  sneeze). 

Hiccough.  —  Lower  the  diaphragm  quickly 
toward  full  inspiration,  and  while  the  lung  is 
expanding  close  the  respiratory  opening  with 
the  moistened  thumb  (hiccough). 

Note  the  sudden  changes  of  pressure  in  the 
two  cavities. 

Perforation  of  the  Pleura.  —  Open  the  inlet  to 
the  pleura. 

Note  the  effect  of  the  opening  into  the  pleural 
cavity  upon  the  lung  and  upon  the  intra-pulmo- 
nary  and  intra-thoracic  pressure. 

Observe  the  result  of  movements  of  the 
diaphragm. 


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